A technique to increase accessibility to late-stage chick embryos for in ovo manipulations

Abstract

Background: During early development, avian embryos are easily accessible in ovo for transplantations and experimental perturbations. However, these qualities of the avian embryonic model rapidly wane shortly after embryonic day (E)4 when the embryo is obscured by extraembryonic membranes, making it difficult to study developmental events that occur at later stages in vivo.

Results: In this study, we describe a multistep method that involves initially windowing eggs at E3, followed by dissecting away extraembryonic membranes at E5 to facilitate embryo accessibility in ovo until later stages of development. The majority of the embryos subjected to this technique remain exposed between E5 and E8, then become gradually displaced by the growing allantois from posterior to anterior regions.

Conclusions: Exposed embryos are viable and compatible with embryological and modern developmental biology techniques including tissue grafting and ablation, gene manipulation by electroporation, and protein expression. This technique opens up new avenues for studying complex cellular interactions during organogenesis and can be further extrapolated to regeneration and stem cell studies.

Fig. 1

A schematic diagram showing treatment of the eggs and embryos at different stages of development. (i–iii) After 3 days of incubation, albumen is withdrawn from the narrow end of the egg, which is then windowed to reveal an intact E3 chick embryo and surrounding vasculature. The eggs are sealed with transparent tape and re-incubated. (iv–vi) On day 5, the tape is cut to reveal the E5 embryo that is partially covered by the CAM. The amnion is dissected and the CAM is displaced away to expose the embryo. The exposed embryo can be manipulated at this stage or re-incubated if later stages are desired. (vii) The exposed embryos do not show any defects and are accessible in ovo between E5–E8 and they can be manipulated and re-incubated until a desired time point.

Fig. 2

Exposure of the E5 chick embryo in ovo. (A–D) Schematics of the embryo viewed from the posterior showing the surrounding amnion (light blue), the chorion (purple) and the vascularized allantois (brown) during membrane dissections. (A’–D’) Bright-field images of the embryo showing the location of the amnion (blue outline) and chorioallantoic membrane (white outline) during membrane dissections. The amnion and chorion are dissected as indicated by the blue and yellow arrows in the regions lateral to the ACM (asterisks). Following the separation of the embryo and surrounding amnion from the CAM (C’), the amnion is peeled away from the cranial region (D’, curved black arrows). The posterior region of the embryo slides out of the amnion and the entire embryo becomes exposed.

Fig. 3

Exposure and survival of embryos at different stages of development. (A) Following membrane dissection, the majority of the embryos are exposed between E5–E7, before they are gradually obscured by the CAM (white dotted line) by E9. (B) The cranial and trunk regions of embryos are accessible for in ovo manipulation between E5–E7. By E9, access to the embryo is limited in most cases to the cranial region. (C) The majority of the exposed embryos are viable between E5–E7 and approximately a third of the embryos are viable during the critical period of organogenesis. The normalized numbers of surviving exposed embryos are reported as a percentage of surviving non-dissected controls at each stage.

Fig. 4

Cell injections into E7 exposed embryos. Cells expressing RCAS-GFP were mixed with food dye and injected as indicated by the arrowheads into (A) the cornea, (B) mesenchyme adjacent to the ear, and (C) mesenchyme of the forelimb.

Fig. 5

Embryo collected 2 days after cell injections showing: (A) Normal development in absence of amnion. Under fluorescence, GFP expression was observed in (B and B’) the mesenchyme adjacent to the ear, (C and C’) the stroma region of the cornea, and (D and D’) the limb mesenchyme adjacent to a presumptive phalangeal bone. Abbreviations: fg-feather germs, et-egg tooth, so-scleral ossicles, ep-epithelium, ststroma, en-endothelium, pb-phalangeal bone. scale bar=100µm