Our ex-ovo/in-ovo procedure is a precise and efficient method for labeling and ablating cells in blastula stage chick embryos that supports development into the fetal period. We have used this procedure to fluorescently label subpopulations of epiblast cells that express myogenic and neurogenic transcription factors with the G8 and E12 MAbs, respectively (4
). In the stage 2 embryo, the G8 and E12 MAbs bind to separate populations of cells in the posterior epiblast () (4
). A second and larger subpopulation of E12-positive cells is present in the anterior epiblast (4
Labeling Embryos Ex-Ovo with Fluorescent Antibodies
Embryos were incubated in host shells in order to analyze the sites of incorporation of G8 and E12 labeled epiblast cells as development progressed. Approximately 75% of embryos were visible through the window in the shell for the first three days after transfer (). All embryos became visible through the window by the fourth day in-ovo, thereby permitting daily observations of morphogenesis. The survival rate of embryos labeled with antibodies during stages 1-4 and returned to host shells and J). E12-positive cells were observable in whole embryos for an additional 48 hours of development by epifluorescence microscopy, although confocal microscopy or sectioning the embryos is required for quantitative analyses. These experiments demonstrate that epiblast cells can be labeled with non-function perturbing antibodies ex-ovo and cultured in host shells without adversely affecting development. Detection of antigens sensitive to endogenous protease digestion may require incubations at 4oC instead of room temperature.
The technique of complement-induced cell lysis was utilized over 50 years ago to examine antigenicity in the chick embryo (28
). We have taken advantage of complement’s ability to lyse cells bound with antibodies to eliminate specific subpopulations within the epiblast that express different cell surface antigens (5
). Incubation in baby rabbit complement alone at a dilution of 1:40 did not increase cell death in the epiblast (). Labeling cells with the G8 and E12 MAbs followed by incubation in complement resulted in lysis of separate populations of epiblast cells, as determined by the uptake of trypan blue () (5
). Testing the specificity of lysis of antibody labeled cells with different concentrations of complement purified from various sources is recommended for each primary antibody and stage of development.
Ablating Cells in the Epiblast with Antibodies and Complement
Lysis of subpopulations of epiblast cells labeled with the G8 or E12 MAbs produced different malformations as development progressed. Elimination of G8-positive cells in the stage 2 epiblast led to a herniation of organs through the ventral body wall (). Loss of integrity of the body wall resulted from a dramatic reduction in skeletal muscle in the myotome (5
). The mechanism whereby G8/MyoD-positive epiblast cells regulate myogenesis in the somites involves the production of the bone morphogenetic inhibitor Noggin (5
). These embryos died between the sixth and seventh day of development.
Lysis of the subpopulation of NeuroM expressing cells recognized by the E12 MAb led to neural tube defects (). The role of E12-positive epiblast cells during neural tube development has not yet been characterized. Embryos treated with E12 and complement remained viable for at least nine days. Exposure to baby rabbit complement alone did not produce malformations in the embryo ().
We have used this ex-ovo/in-ovo procedure to follow the fate of cells labeled in the epiblast and to determine the effects of ablating them on differentiation and morphogenesis (5
). Our procedure requires no special equipment other than a dissecting microscope and a fiber optic light source for illumination from above. A significant advantage of this ex-ovo/in-ovo procedure over previously published culture methods, including the technique of manipulating embryos through a window in the shell, is that experiments can be initiated prior to the onset of gastrulation and subsequently grown to fetal stages with a high rate of survival. The application of antibodies for labeling or ablation is achieved without perturbing the embryo.
The procedures we have developed for cell tracking and ablation depend on the existence of antibodies that recognize cell surface antigens expressed on specific subpopulations in the early embryo. Other methods of marking cells that could be used with our ex-ovo/in-ovo system include transplantation of quail cells into chick hosts (29
), microinjection of vital dyes (30
) and microinjection or electroporation of genes coding for fluorescent proteins (34
). Microinjection of fluorescent dyes followed by laser ablation was used to eliminate cells in stages 12-18 embryos grown in shell-less cultures (17
). This approach could be attempted with blastula stage embryos using the ex-ovo/in-ovo protocol.
An important advantage of our ex-ovo/in-ovo method is that it supports the development of embryos manipulated at the blastula stage into the fetal period. The success of this method may be attributed, in part, to returning the embryo to the shell that maintains the appropriate tension on the yolk and provides calcium to the embryo. Although we have not yet extended our experiments beyond day 17, the host shell is expected to support development until hatching.