The HUDSEN Atlas: a three-dimensional (3D) spatial framework for studying gene expression in the developing human brain

Abstract

We are developing a three-dimensional (3D) atlas of the human embryonic brain using anatomical landmarks and gene expression data to define major subdivisions through 12 stages of development [Carnegie Stages (CS) 12-23; approximately 26-56 days post conception (dpc)]. Virtual 3D anatomical models are generated from intact specimens using optical projection tomography (OPT). Using MAPAINT software, selected gene expression data, gathered using standard methods of in situ hybridization and immunohistochemistry, are mapped to a representative 3D model for each chosen Carnegie stage. In these models, anatomical domains, defined on the basis of morphological landmarks and comparative knowledge of expression patterns in vertebrates, are linked to a developmental neuroanatomic ontology. Human gene expression patterns for genes with characteristic expression in different vertebrates (e.g. PAX6, GAD65 and OLIG2) are being used to confirm and/or refine the human anatomical domain boundaries. We have also developed interpolation software that digitally generates a full domain from partial data. Currently, the 3D models and a preliminary set of anatomical domains and ontology are available on the atlas pages along with gene expression data from approximately 100 genes in the HUDSEN Human Spatial Gene Expression Database (http://www.hudsen.org). The aim is that full 3D data will be generated from expression data used to define a more detailed set of anatomical domains linked to a more advanced anatomy ontology and all of these will be available online, contributing to the long-term goal of the atlas, which is to help maximize the effective use and dissemination of data wherever it is generated.

Fig. 1

Painted anatomical domains in 2D and 3D. This shows the subdivisions of the central nervous system that have been defined in the CS22 model. The domains can be visualized in 2D sections (A) or in 3D (B), and are linked to the ontology shown in (D). The colour scheme is indicated in the key (C).

Fig. 2

Comparison of painted domains with gene expression data. This shows data from immunohistochemical experiments at CS22. Each image was captured at a resolution of 8.2 μm/pixel. PAX6 (A,E) has been compared with GAD65 (B) and OLIG2 (F), at 2 levels through the head of a CS22 embryo [the section plane is indicated in the sagittal views (D,H)]. When digital sections from the painted OPT model (C,G) are aligned with the experimental sections it becomes evident that the gene expression patterns define boundaries between different anatomical regions. Both PAX6 (A) and GAD65 (B) are strongly expressed in the pallium (red) and define the boundary between the pallium and subpallium (orange). Both genes are also strongly expressed in Prosomere 3 (green), stopping at the boundary with the hypothalamus [painted in brown, the boundary indicated with a white arrow in (A) and (B)]. PAX6 is more weakly expressed within the striatal subpallium, allowing the boundary between the medial and lateral ganglionic eminences [white arrowhead in (A)] to be distinguished. At a slightly more caudal level, PAX6 (E) has been compared with OLIG2 (F). At this section level PAX6 is expressed strongly in both the pallium and hypothalamus, but negative in the subpallium. In both areas the expression stops sharply at the subpallial boundary (black arrowheads). PAX6 also defines the alar/basal boundary (black arrow) within rhombomere 1 (painted purple) in the hindbrain. In contras, OLIG2 is expressed strongly in the subpallium, but is negative in the pallium.

Fig. 3

3D OLIG2 expression domain at CS19. A 3D gene expression pattern for OLIG2 in the forebrain of the CS19 model was generated by mapping a series of 2D immunohistochemical sections, an example of which is shown in (A). This mapping is carried out section by section to incrementally build up the 3D expression pattern [shown in red in (B)]. The original section plane (E,F) is indicated on the sagittal view (D). Note that when the angle of section is changed [indicated on the sagittal view in (G)] the domain is displayed as a series of lines where the section plane intersects the individual 2D domains (H). As described above, the 3D-IDM software tracks and matches components in individual sections and interpolates the expression patterns on the intervening sections, generating a smooth 3D domain that can be cut, checked and refined in any plane. The interpolated 3D domain for our example is shown in cyan in (C), the spaces between the sections having been ‘filled in’, and the domain no longer appears as a series of lines on the 2D section, but as a continuous area (I), as in the original section plane (F).

Fig. 4

The HUDSEN gene expression database. (A) Home page of the HUDSEN database website (http://www.HUDSEN.org) displaying a table of the genes currently contained in the database. (B) The results of a search for PAX6. The database can be queried in a number of ways: by Anatomical name, Carnegie Stage, Gene or Probe ID. Alternatively, the entire contents of the database can be displayed for browsing. (C) Screenshot of an entry in the HUDSEN Database showing PAX6 expression at CS22. Thumbnail images of the experimental sections and the expression mapped onto the OPT sections can be clicked on to provide full resolution images. A 3D view showing the 3D context of the mapped data is also provided, which can be downloaded as a movie. Where gene expression intersects with painted anatomical domains, percentage data is shown, providing both relative quantitative and qualitative information about the sites of the gene expression. In addition, probe/antibody information is provided, together with any related publications.