Coronal Sections

David J. Price1, Elizabeth Graham2, Julie Moss2, Chris Armit2 and Richard Baldock2,    1Centre for Integrative Physiology, The University of Edinburgh, Edinburgh, UK,    2MRC Human Genetics Unit, IGMM, The University of Edinburgh, Edinburgh, UK

The original The Atlas of Mouse Development by Matt Kaufman (MK) included transverse and sagittal sections at multiple stages of development, but only two plates of coronal sections (stages E14.5 (plate 34) and E16.5 (plate 39)). When a revised version of the Atlas was suggested, a survey of users recommended that the series of coronal sections should be extended to include additional stages of development, particularly the brain at E11, E11.5, E12.5, E13.5, and E15.5. This chapter presents these new coronal sections, together with some new sections to extend the E14.5 images provided in the original Atlas. These sections include some original annotations from MK that are supplemented by more detailed brain annotations.

Keywords

Atlas; ontology; Matt Kaufman; mouse development; histology plates; coronal sections

Introduction

The Atlas of Mouse Development by Matt Kaufman (MK; Kaufman, 1994) is the defining standard for mouse development and remains an essential text for developmental biologists and biomedical scientists. The Atlas has a series of plates of histological sections through embryos that have been selected to represent each Theiler stage of development. Each image within a plate is carefully annotated with the tissues visible in each section clearly labeled. The original Atlas includes two embryos sectioned coronally at E14.5 and E16.5. When users were asked what should be added to a revised edition of the Atlas, there was a clear demand for additional coronal sections to complete the developmental series in order to make the development of the embryonic brain easier for the research community to understand.

This chapter provides these new coronal sections as a series of plates that can be used in conjunction with the original Atlas and the chapters of this volume. Before ceasing work on the revised Atlas (now presented as this Atlas Supplement), MK selected the embryos, specified the sections that were most illustrative and should be used for these new plates, and provided some preliminary annotation. Here, we have taken that selection and extended the annotation.

The printed version of these sections follows the style of the Atlas with each image labeled with lines and numbers, with each number linked to an annotation on the same two-page view. These coronal sections only show the head-region of the section so that the brain is printed at the highest possible resolution. We also provide the fully annotated, high-resolution images online within a "zoom" style viewer (see www.emouseatlas.org).

While MK's annotations in the original Atlas were given as short descriptions, the plates here use the anatomical terms defined by the Edinburgh Mouse Atlas Project (EMAP) mouse developmental anatomy ontology (see Chapter 23 and Hayamizu et al., 2013). This ontology uses a controlled vocabulary, and this has been extended to include some new annotations shown here. An advantage of using the EMAP ontology descriptions for labeling tissues is that the online visualization of each section is directly linked (through the hidden ontology term IDs) to other database resources, and this allows a user to, for example, directly query gene-expression in any anatomical component. The EMAP ontology also describes the stage range for specific anatomical components, and this allows researchers to follow, in order of appearance, the various anatomical components of the developing mouse embryo. We hope that the use of EMAP ontology terms will allow researchers to explore embryonic development in new ways.

Methods

To generate the new coronal sections, we used C57Bl6J×CBA F1 embryos that were collected, fixed, and washed in the same way as those for The Atlas of Mouse Development (Kaufman, 1994). Embryos were dehydrated through an ethanol gradient, cleared in xylene, then infiltrated and embedded in paraffin wax (56°C melting point). They were then embedded on their side on a layer of setting wax in molds of suitable size, and this allowed the embryo to be oriented for coronal sectioning. The trimmed wax block was mounted on a wooden chuck and attached to the microtome (Leica RM2264 motorized with retraction and microscope attachment). Starting at the front of the embryo, serial sections were cut at 7 µm, using the motorized setting, directly onto distilled water (room temperature) in a trough specifically designed to keep section loss to a minimum (this was manufactured "in house" to fit the disposable knife holder).

Strings of sections were removed from the trough on a glass slide and carefully introduced into a floating-out bath at 42-45°C where they were allowed to stretch out for a few seconds. Time and temperature were carefully monitored to make the stretching consistent. The sections were then aligned on a cleaned glass slide, withdrawn from the bath, and dried vertically at room temperature overnight. Before staining, the slides were placed in an oven at 60°C for 2 h, dewaxed in xylene, and rehydrated through an ethanol gradient to water. The sections were stained in Mayer's Haemalum for 5 min, and aqueous Eosin Y for 3 min, then dehydrated, cleared in xylene, and mounted in DPX (coverslip thickness No. 1.5: 0.13-0.17 mm).

Once the mountant had completely set, the slides were digitized using the Olympus DotSlide, which capture a complete image of each section by automated scanning over the area of tissue. To optimize the digital image focus, we manually set focus points for each section then the scanning was performed with either a ×10 or a ×20 objective resulting in image pixel resolutions of 0.32 and 0.16 µm, respectively.

The DotSlide images were output to "tiff format" and converted to multiresolution tiled "pyramidal" format using the VIPS image processing software (Martinez and Cupitt, 2005). This provides the required tiled format for a zoom-viewer interface using the IIP3D server (Husz et al., 2012). In addition, we developed a bespoke MySQL database to hold the plate information and annotations. The interface used to deliver the coronal image can be configured to allow editorial placement of points associated with any ontology term.

Results

The following table lists the stages covered by these additional coronal sections and some additional data for each embryo.

Discussion

These coronal sections extend the series of coronal sections provided in the original The Atlas of Mouse Development. Low-resolution images are available in the Supplement, while high-resolution versions are available from the eMouseAtlas Web site (www.emouseatlas.org) and these show the annotations identified by the matching number in the plates shown here. The same images available at much higher resolution on the Internet are an open-access community resource available freely for scientific and educational purposes. In this context, we hope to invite expert developmental anatomists to contribute to the resource to enhance the annotations available and so increase our knowledge of developmental anatomy.

Acknowledgments

We thank Allyson Ross for preparing and sectioning the embryos and Nick Burton for developing the editing viewer that enabled anatomical terms to be selected and annotations to be placed on images.

References

1. Hayamizu TF, Wicks MN, Davidson DR, Burger A, Ringwald M, Baldock RA. EMAP/EMAPA ontology of mouse developmental anatomy: 2013 update. J Biomed Semantics. 2013;4:15 In: http://dx.doi.org/10.1186/2041-1480-4-15.

2. Husz ZL, Burton N, Hill W, Milaev N, Baldock RA. Web tools for large-scale 3D biological images and atlases. BMC Bioinformatics. 2012;13:122 In: http://dx.doi.org/10.1186/1471-2105-13-122.

3. Kaufman MH. The Atlas of Mouse Development revised ed. London: Academic Press; 1994.

4. Martinez, K., Cupitt, J., 2005. VIPS-a highly tuned image processing software architecture. In: Conference Proceedings, IEEE International Conference on Image Processing 2, Genova, pp....