Tracking and ablating subpopulations of epiblast cells in the chick embryo

Abstract

The early chick embryo contains subpopulations of cells that express lineage-specific transcription factors. We have developed protocols to examine the role of these cells during development that involve labeling them for cell tracking purposes and ablating them within the epiblast. The procedures take advantage of the fact that subpopulations of epiblast cells differentially express cell surface antigens recognized by monoclonal antibodies. Embryos are removed from the shell and incubated on the yolk with an antibody. Cells that bind the antibody are either tagged with a fluorescent secondary antibody or lysed with complement. For long-term analyses, embryos are returned to a host shell and placed in an incubator. This method of whole embryo manipulation ex-ovo and incubation in-ovo supports normal development into the fetal period.

Keywords: Chick Embryo; Staining and Labeling.

Fig. 1. Labeling Embryos Ex-Ovo with Fluorescent Antibodies

Stage 2 embryos were labeled with the G8 and E12 MAbs and fluorescent secondary antibodies (red). Nuclei were stained with Hoechst dye (blue). The inset in panels A and B illustrates the location of cells labeled with the G8 (A) and E12 MAbs (B) in the posterior epiblast of unsectioned stage 2 embryos. Labeled embryos developed normally after returning them to the shell and incubating them for three (C), five (D), or 13 days (E). The region of the somite outlined in the hematoxylin and eosin stained section of the 5 day embryo (F) is shown at higher magnification in the fluorescence photomicrograph (G). Epiblast cells stained with the G8 MAb were incorporated into the dorsomedial region of the dermomyotome and myotome (G). The locations of epiblast cells labeled with the E12 MAb at stage 4 are outlined in the drawing of the unsectioned stage 14 embryo (H). Cells stained with E12 were visible in the brain (I) and neural tube (J). Bar = 9 µm in A, B and C and 54 µM in F-I.

Fig. 2. Ablating Cells in the Epiblast with Antibodies and Complement

Stage 2 embryos were incubated with PBS (CT), the G8 or E12 MAb and treated with complement. Lysed cells were visible in the epiblast after staining with trypan blue (arrows). Only a few dead cells were visible in the epiblast after incubation with PBS and complement (arrows in A). The inset in panel A illustrates the location of lysed cells in panels B and C. Subpopulations of dead cells were visible in the posterior epiblast after lysing cells bound with the G8 (B) and E12 (C) MAbs. Six days after treatment, embryos treated with complement alone appeared normal upon gross inspection (D) and in sections stained with hematoxylin and eosin (G). Ablation of G8 labeled cells resulted in herniation of organs through the ventral body wall (E and H). Incubation with E12 and complement resulted in neural tube (nt) defects visible in tissue sections (I). Bar = 135 µM in A-C and G-I.

Fig. 3. Culturing Embryos in Host Shells after Ex-Ovo Manipulation.

Embryos on the yolk were placed in a Petri dish. Prior to manipulation, excess albumen was teased away from each side of the embryo with a Kimwipe (A). A solution was delivered to the lateral edge of the embryo with a pipet (B). A piece of parafilm was used to spread the solution over the embryo (B and C). Host shells were prepared by cutting an opening in the top of the shell large enough for the yolk to fit through (D and E). The thick albumen and embryo on the yolk were poured into the empty host shell (F). Thin albumen was added to approximately 0.5 cm below the opening in the shell (G). The opening was covered with plastic wrap (H).