A new method to transfect the hypoblast of the chick embryo reveals conservation of the regulation of an Otx2 enhancer between mouse and chick extraembryonic endoderm

Abstract

Background: The mouse anterior visceral endoderm (AVE) and the chick hypoblast are thought to have homologous roles in the early stages of neural induction and primitive streak formation. In mouse, many regulatory elements directing gene expression to the AVE have been identified. However, there is no technique to introduce DNA into the chick hypoblast that would enable a comparison of their activity and this has hampered a direct comparison of the regulation of gene expression in the mouse and chick extraembryonic endoderm.

Results: Here we describe a new method to introduce DNA into the chick hypoblast, using lipofectamine-mediated transfection. We show that the hypoblast can be easily transfected and that it starts to express a luciferase reporter within 2 hours of transfection. The validity of technique is tested by following the movement and fate of hypoblast cells, which reveals their translocation to the anterior germinal crescent. We then introduce a vector containing GFP driven by the mouse VEcis-Otx2 enhancer (which directs gene expression to the mouse AVE) and we detect activity in the hypoblast.

Conclusion: The new technique for delivering expression constructs to the chick hypoblast will enable studies on gene activity and regulation to be performed in this tissue, which has proved difficult to transfect by electroporation. Our findings also reveal that regulatory elements that direct gene expression to the mouse AVE are active in chick hypoblast, supporting the idea that these two tissues have homologous functions.

Figure 1

Transfection of the hypoblast. A-E: pCAB-Luc monitored by luciferase detection at time points following hypoblast tranfection: 2 hours (A), 5 hours (B), 7 hours (C), 10 hours (D), 14 hours (E). Coloured pixels represent light intensity with blue being least intense and red most intense. F-M: GFP expression in whole embryo- (F,G) and epiblast-transfected (H,I) tissues (fluorescence images, F, H and overlaid with brightfield images, G, I). Arrowheads indicate cells expressing GFP in H. J-M: dsRed and GFP expression in the hypoblast 8 hours (J,K) and 14 hours (L,M) after transfection. An overlay of fluorescence and brightfield images are shown in K and M. N-Q: longer culture periods reveal the transfected hypoblast (shown here with GFP only) in stage HH5 (N), HH7 (O), HH8- (P) and HH9 (Q) embryos. Arrows indicate labelled cells in the yolk sac. R-S: VEcis-Otx2 driving GFP is detected in transfected hypoblasts after 7–8 hours (R) and is seen in the crescent-shaped hypoblast at stage 4+/5 (S). T-U: Co-electroporation of VEcis-Otx2-GFP and CMV-dsRed shows GFP localised in the embryo in the rostral to the hindbrain (T) and also occasionally in the hindbrain (U) (arrows). dsRed is expressed in the entire electroporated region and is seen more caudally in the embryo and in the area opaca (arrowhead).