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- RESULTS AND DISCUSSION
- EXPERIMENTAL PROCEDURES
The chick embryo is a valuable model organism used to study vertebrate development. Besides the availability and low cost of fertilized eggs, early-stage chick embryos are readily accessible in ovo for visualization and experimental manipulations. During the initial 3 days of incubation, a chick embryo, which begins as a blastodisk, undergoes gastrulation, neurulation, and several morphogenetic movements to form distinct cranial and trunk structures. Within this period of development, the embryos can be easily accessed and manipulated at different developmental time points by “windowing” the egg-shell, then re-incubating until the desired late embryonic stages. Using this approach, several techniques such as DiI injections (del Barrio and Nieto, 2002) and tissue transplantation (Le Douarin, 1973; Goldstein, 2006; Lwigale and Schneider, 2008) or tissue ablation (Summerbell, 1974; Bénazéraf et al., 2010) have been extensively utilized to study cell fate and pattern formation. In addition, gain- and loss-of-function studies involving electroporation of DNA, RNAi, morpholinos, and virus constructs are now widely used in molecular studies involving chick embryos (see reviews by Nakamura, et al., 2004; Sauka-Spengler and Barembaum, 2008).
Despite their versatility as model organisms for studying developmental biology, chick embryos are rapidly surrounded by extraembryonic membranes, making it difficult for experimental manipulations in ovo past embryonic day (E)5. The chick embryo is initially covered by a transparent vitelline membrane that separates it from the albumen. Beginning at about HH stage 12 (Hamburger and Hamilton, 1951), the amnion and chorion membranes overlap the forebrain and tail bud by growing towards each other and fuse to cover the entire embryo by stage 18. At about this time another membrane, the allantois, forms as a balloon-like structure in the hind-gut region of the embryo. The allantois fuses with the chorion to form the chorioallantoic membrane, which stores nitrogenous waste and is involved in respiration and calcium transport. The chorioallantoic membrane grows rapidly and covers most of the embryo by stage 20 (E5). During early development, embryos can be easily accessed through the vitelline membrane, or chorion, and by dissecting the amniotic membrane. However, accessing an E5 or older embryo through the overlying chorioallantoic membrane is lethal. Therefore, most developmental biology studies using chick embryos in ovo are limited to relatively early stages of development, prior to critical periods of organogenesis that may require different signals and cellular interactions.
Several genes associated with cellular interactions and differentiation during organogenesis of the eye, ear, brain, skin, and tissues such as bones and cartilages, are either transiently expressed or initiate expression during later stages of embryogenesis. To increase accessibility to later stages (older than E4), methods such as ex ovo culture of chick embryos have been developed (New, 1955; Auerbach et al., 1974; Dugan et al., 1991; Datar and Bhonde, 2005; El-Ghali et al., 2010). While these methods overcome the complications that arise from adhering of the embryo to the eggshell and increase access to the embryo, the chorioallantoic membranes expand and obscure the embryo from manipulation at later stages. In addition, chick embryos cultured ex ovo show severe retardation in growth probably due to Ca2+ deficiency, which is naturally provided by the eggshell (Luo and Redies, 2005).
Until now, there have been no attempts to address the obstacles that impede manipulation of the late-stage chick embryos in ovo. Current studies of organogenesis can only be conducted in vitro on tissue that is isolated from the influence of the endogenous embryonic environment, which may affect gene expression and crucial cellular interactions. In this study, we report a novel method that further strengthens the chick embryo as a model for developmental biology. This stepwise method combines preparation of fertilized chick eggs at different developmental time points with careful dissection and relocation of extraembryonic membranes to increase accessibility to embryos at late stages of development. Embryos exposed using this technique are viable and easily accessible in ovo for manipulation of tissues during organogenesis. We show that different regions of the exposed E7 embryos are easily accessible for manipulations such as injection with RCAS-GFP-expressing cells. After two days of re-incubation, the injected cells can be tracked by monitoring GFP expression in various tissues. Late-stage embryos exposed by this technique can be manipulated using standard developmental approaches to study gene function, cellular interactions, tissue regeneration, and stem cell potential during organogenesis.