The AERO system: a 3D-like approach for recording gene expression patterns in the whole mouse embryo

Abstract

We have recently constructed a web-based database of gene expression in the mouse whole embryo, EMBRYS (http://embrys.jp/embrys/html/MainMenu.html). To allow examination of gene expression patterns to the fullest extent possible, this database provides both photo images and annotation data. However, since embryos develop via an intricate process of morphogenesis, it would be of great value to track embryonic gene expression from a three dimensional perspective. In fact, several methods have been developed to achieve this goal, but highly laborious procedures and specific operational skills are generally required. We utilized a novel microscopic technique that enables the easy capture of rotational, 3D-like images of the whole embryo. In this method, a rotary head equipped with two mirrors that are designed to obtain an image tilted at 45 degrees to the microscope stage captures serial images at 2-degree intervals. By a simple operation, 180 images are automatically collected. These 2D images obtained at multiple angles are then used to reconstruct 3D-like images, termed AERO images. By means of this system, over 800 AERO images of 191 gene expression patterns were captured. These images can be easily rotated on the computer screen using the EMBRYS database so that researchers can view an entire embryo by a virtual viewing on a computer screen in an unbiased or non-predetermined manner. The advantages afforded by this approach make it especially useful for generating data viewed in public databases.

Figure 1. Overall setup of the AERO system hardware.

(A) HiRox microscopy and (B) schematic representation of the rotary head. A specimen is placed on an agarose layer prepared in a cylindrical container or petri-dish. The key feature of this component is the two mirrors installed in the rotary head, which allows the camera to capture an image that is tilted 45 degrees to the microscopic stage. The rotary head is rotated by 360 degrees while capturing images at 2-degree intervals. Therefore the 180 serial images are captured in one simple operation. After image acquisition, these serial images are automatically arranged so as to produce a movie.

Figure 2. AERO images of Pax1 and Aes.

Expression of Pax1 (paired box gene 1) and Aes (amino-terminal enhancer of split) in the E11.5 mouse embryo was visualized by in situ hybridization. (A) Left- and Right-side AERO images of Pax1. The expression in the somite and maxillary process is observed. (B)Three AERO images of Aes. The Left-, Front- and Back-side AERO images cover almost all the body parts of the embryo. The expression in the brain, somite, forelimb and hindlimb is clearly observed from various angles.

Figure 3. Comparison of conventional 2D images and AERO images.

The expression of Lmx1b, Isl1 and Cited1 at E11.5 is visualized by in situ hybridization. The conventional 2D images (A, C, E) from the EMBRYS database and the AERO images (B, D, F). (A, B) The AERO images reveal Lmx1b expression is restricted to the dorsal side and is absent on the ventral side of the forelimb-bud (arrow) and hindlimb bud (arrow head). D:dorsal side, V: ventral side. (C, D) Isl1 expression in the genitalia is clearly observed in the AERO images (red arrows). (E, F) Cited1 expression in the maxillofacial region is more clearly observed in the AERO images than 2D image (red arrows).