Hatchability of chicken embryos following somite manipulation.

The avian embryo has been a classical model to study early development because the embryo is easily accessible for manipulation of embryonic cells and structures. The usefulness of the chicken embryo increased with the development of quail-chick transplantation techniques (1,2). The ability to transplant Japanese quail cells into a chicken embryo provides a method to trace the developmental fate of the implanted quail cells in a chicken host embryo because the dense magentacolored heterochromatin and nucleoli of the Japanese quail nuclei in Feuglenstained histological sections distinguishes donor quail nuclei from host chicken nuclei. The quail nucleolar marker is heritable, making it possible to determine the ultimate developmental fate of quail cells transplanted into host chicken embryos. Furthermore, monoclonal antibodies have been developed that recognize quail nuclei, but they do not recognize chicken nuclei (QCPN; Developmental Studies Hybridoma Bank, Iowa City, IA, USA). Therefore, it is possible to distinguish quail cells from chicken cells in a quailchick chimera using classical histochemistry (Feuglen staining) (1–3) or immunohistochemistry (4–6). Although the quail-chick chimera has been a powerful tool for developmental biology research, it would be better to employ a chicken/chicken implantation system because it would eliminate any potentially confounding species-specific effects on any experiments. However, it has been previously impossible to employ a chicken/chicken system to study cell fate because there have not been any previously reported useful lines of transgenic chickens expressing histologically convenient reporter genes. Lines of transgenic chickens carrying the Escherichia coli lacZ reporter gene and expressing β-galactosidase have recently been developed (7), making it possible to follow the developmental fate of transgenic chicken cells implanted into wild-type embryos. Therefore, a new tool for developmental biology research that is a significant improvement over the quail-chicken system is now available. A potential difficulty in avian embryonic developmental biology research is the ability to follow embryonic cell fate through hatching and adult maturity. Few researchers have attempted to study the effect of embryonic manipulation on post-hatch chickens. In one case, a portion of the neural tube and the somites was removed from 621 chicken embryos and replaced with the same portion of the neural tube from quail embryos, but only 46 somatic chimeras hatched (7%). Seventeen of the 46 somatic chimeras exhibited various abnormalities in the limbs at hatching. Therefore, only 29 hatched somatic chimeras from the original 621 manipulated embryos were fully viable at hatch (5%) (8). The surviving quailchick chimeras appeared normal at hatch but died after a few weeks of age because of an interspecies-related demylenation of the spinal cord (9). It appears that the quail-chicken system is not applicable for studying the effects of embryonic manipulations on adult birds. Furthermore, chick-chick neural tube/somite chimeras also resulted in a very low hatchability (2 out of 40; 5%) (8). Therefore, it also appears that it is difficult to achieve a reasonable level of hatchability following embryonic manipulations to study post-hatch development. It is important to note that these experiments (8,9) included an invasive transfer of the spinal cord between embryos. However, other less invasive manipulations have also resulted in relatively low hatchability. For example, primordial germ cells were injected into the dorsal aorta of recipient stage 15 (10) embryos, and only 7%–14% of the injected embryos hatched in one study (11), and approximately 26% of the injected embryos hatched in a second study (12). Furthermore, Naito et al. (11,12) used a laborious surrogate eggshell culturing procedure for their studies, and the procedures reported in the present study employ a simple eggshell windowing technique. For the most part, hatchability data for somatic manipulations do not appear readily available in the scientific literature. Therefore, the objective of this manuscript is to report our procedures for the manipulation of embryonic chick somites and the associated level of hatchability that was achieved using the reported procedures. The rationale was to demonstrate the results of microinjection and the level of hatchability associated with the experimental manipulations. Future studies will focus on implanted cell fate. The experimental procedures are useful for studies aimed at understanding the effect of embryonic manipulations on post-hatch development. Freshly laid eggs were placed into an incubator until they reached stages 10–15 (10). Fertile eggs were stored with the blunt end up for 2–3 h. Subsequently, a hole was cut in the shell on the blunt end of the egg with surgical scissors (Figure 1). The embryos were visualized in the egg using lateral illumination though a wratten 47 blue gelatin filter (Sigma, St. Louis, MO, USA). The blue filter visualizes the somites in the embryo while it is still in the egg (Figure 2). Therefore, it is possible with blue light illumination to manipulate the chicken embryos without traditional India ink staining. All embryos were manipulated between approximately stage 10 and stage 15 (10). Figure 3 also demonstrates that it is possible to sufficiently visualize the somites and other structures for implantation studies without India ink staining because 3 μL of a 50 μg/mL solution of propidium iodide in 0.1% SDS were successfully injected into a somite. Subsequently, the injected embryo was fixed with 4% paraBenchmarks