3 dimensional modelling of early human brain development using optical projection tomography

Abstract

Background: As development proceeds the human embryo attains an ever more complex three dimensional (3D) structure. Analyzing the gene expression patterns that underlie these changes and interpreting their significance depends on identifying the anatomical structures to which they map and following these patterns in developing 3D structures over time. The difficulty of this task greatly increases as more gene expression patterns are added, particularly in organs with complex 3D structures such as the brain. Optical Projection Tomography (OPT) is a new technology which has been developed for rapidly generating digital 3D models of intact specimens. We have assessed the resolution of unstained neuronal structures within a Carnegie Stage (CS)17 OPT model and tested its use as a framework onto which anatomical structures can be defined and gene expression data mapped.

Results: Resolution of the OPT models was assessed by comparison of digital sections with physical sections stained, either with haematoxylin and eosin (H&E) or by immunocytochemistry for GAP43 or PAX6, to identify specific anatomical features. Despite the 3D models being of unstained tissue, peripheral nervous system structures from the trigeminal ganglion (approximately 300 microm by approximately 150 microm) to the rootlets of cranial nerve XII (approximately 20 microm in diameter) were clearly identifiable, as were structures in the developing neural tube such as the zona limitans intrathalamica (core is approximately 30 microm thick). Fourteen anatomical domains have been identified and visualised within the CS17 model. Two 3D gene expression domains, known to be defined by Pax6 expression in the mouse, were clearly visible when PAX6 data from 2D sections were mapped to the CS17 model. The feasibility of applying the OPT technology to all stages from CS12 to CS23, which encompasses the major period of organogenesis for the human developing central nervous system, was successfully demonstrated.

Conclusion: In the CS17 model considerable detail is visible within the developing nervous system at a minimum resolution of approximately 20 microm and 3D anatomical and gene expression domains can be defined and visualised successfully. The OPT models and accompanying technologies for manipulating them provide a powerful approach to visualising and analysing gene expression and morphology during early human brain development.

Figure 1

CS17 OPT model (a). still shot from movie of 3D OPT model of a CS17 human embryo (approximately 41 days of development). bv, blood vessel; drg, dorsal root ganglion; h, heart; H, hindbrain; l, liver; T, telencephalon; v, vertebrae. (b; Additional file 1) Mpeg movie of 3D CS17 OPT model.

Figure 2

Comparison of digital OPT sections with histology sections from the same embryo Digital OPT sections of the CS17 model (a, d and g), viewed using MAPaint software, compared with sections stained using antibodies against GAP43 (b, e and h), and Haematoxylin and Eosin stained sections (c, f and i). In b, e and h, expression is demonstrated by the brown chromagen. Structures of 20 μm in diameter (for example the hypoglossal rootlets) are clearly identifiable, as are the differences amongst a variety of developing tissues. bv, blood vessel; D, diencephalon; drg, dorsal root ganglion (~150 μm); fp, floor plate; H, hindbrain; III, oculomotor nerve; M, midbrain; nh, neurohypophysis (~200 μm by ~50 μm); ov, otic vesicle; r, collapsed roof of 4th ventricle; sc, spinal cord; Vg, trigeminal ganglion (~300 μm by ~150 μm); vl, ventricular layer; X, vagus nerve; Xg, vagus ganglion; XIIroot, hypoglossal rootlets (~20 μm); zli; zona limitans intrathalamica (~100 μm, core is ~30 μm). Scale bars = 200 μm

Figure 3

Painted anatomical domains. Fourteen regions of the central nervous system in the CS17 specimen have been defined and painted. Forebrain, red (secondary prosencephalon), dark orange (prosomere 3 including ventral thalamus), light orange (prosomere 2 including dorsal thalamus) and yellow (prosomere 1 including pretectum); midbrain, light green; hindbrain; isthmus, dark green; various shades of blue and purple indicate rhombomeres 1–6 and the caudal medulla oblongata; spinal cord, dark red. (a; Additional file 2) In the Mpeg movie sagittal and transverse views of the painted model are shown, together with a representation of the 3D domains. The model is first sectioned in the transverse plane. This section plane has been matched to that of the histology sections shown in fig 2. As the section is moved through the model the corresponding position is displayed in the 3D box, and by a line on the sagittal section. The model is then moved through the sagittal plane, and the position shown by a line on the transverse section. A snapshot of the fourteen 3D anatomical domains (b), and two examples of painted sections that intersect several anatomic domains (i.e., are topologically nearly horizontal to the reconstructed transverse boundaries) (c). The position of the two digital transverse sections is indicated by white lines on the 3D view.

Figure 4

CS17 OPT model showing 3D gene expression domains. (a) Digital sagittal section through the CS17 OPT model, with the GAP43 gene expression domain shown in red. The plane of this sagittal section is shown by a line on the corresponding transverse section in (b). The GAP43-negative region in the hindbrain floor plate is shown on both sections by an arrow. (c) The same digital sagittal section, with PAX6 gene expression displayed in green. (d) High power images of GAP43 and PAX6 expression near the diencephalon/midbrain boundary. The upper two panels correspond to the rostral midbrain, where there is no expression of GAP43 or PAX6. The lower two panels correspond to the caudal diencephalon, in the region of the posterior commissure. Here both genes are expressed (brown chromagen).

Figure 5

3 dimensional gene expression domains. A surface rendered model of the 3D expression pattern of PAX6. Separate gene expression domains in the forebrain and hindbrain are shown in green. For reference the neural tube has been painted pale grey and the eye dark grey. The diencephalon/midbrain (D/M) boundary, the absence of staining in the zona limitans intrathalamica (zli), plus the forebrain alar-basal boundary and the striatopallidal boundary in the basal telencephalon can be seen by viewing the 3D model at various angles (a, frontal and b, lateral). (c; Additional file 3) Mpeg movie of the PAX6 expression domain.

Figure 6

OPT models of CS12 to CS23. Still shots of the left lateral side of 3 dimensional OPT models of human embryos spanning the major period of organogenesis (CS12-CS23). The developmental stage (e.g. CS12), specimen number (e.g. N285) and karyotype for each model are given underneath. The movies for all of these models can be viewed at [27]. The full models for all stages are available on request.