3 dimensional modelling of early human brain development using optical projection tomography
© Kerwin et al; licensee BioMed Central Ltd. 2004
Received: 26 April 2004
Accepted: 06 August 2004
Published: 06 August 2004
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.
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 (~300 μm by ~150 μm) to the rootlets of cranial nerve XII (~20 μm in diameter) were clearly identifiable, as were structures in the developing neural tube such as the zona limitans intrathalamica (core is ~30 μm 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.
In the CS17 model considerable detail is visible within the developing nervous system at a minimum resolution of ~20 μm 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.
Brain development, particularly in human, involves complex changes in shape and structure over time. During a period of approximately 4 weeks (from 26 to 56 days of development; Carnegie stages CS12 to CS23) the major subregions of the human brain are established and development proceeds from a simple neuroepithelial tube to a highly complex three dimensional (3D) structure . For many years it has been recognized that three dimensional models are an important aid to interpreting these developmental changes. In the past, these have been based on labour intensive methods for reconstructing representations of physical sections (e.g. Born reconstructions ) or, more recently, on computer-based methods, although these are still labour intensive . Non-invasive techniques have also been used and these have advantages of speed and lack of sectioning artifacts and, for example with MRI, the ability to generate in vivo images and good quality images for larger specimens . Recently, a new, rapid and non-invasive 3D modelling method, Optical Projection Tomography (OPT; [5, 6]), has become available, and we have used this method to generate computer-based 3D models from intact early human developmental specimens. OPT has the advantage over MRI in that detailed models can be produced from small samples. With MRI, low signal-to noise ratios make it more difficult to obtain high quality data from embryos younger than CS17 . MRI is likely to be useful for specimens larger than CS23 where the size of the specimen and the density of the tissue are too great to allow penetration of the light.
The OPT models are visualised and manipulated using MAPaint, a suite of software programmes developed as part of the Edinburgh Mouse Atlas Project (http://genex.hgu.mrc.ac.uk[8–10]). The software allows any OPT model to be digitally sectioned in any plane and several different planes can be viewed simultaneously. These planes can be selected at any arbitrary viewing orientation and position through the model. In addition anatomical regions can be defined and manually "painted", allowing the user to interactively assess developing anatomy.
OPT reconstructions were made of human embryos for a number of different stages of development. In all cases, reconstructions were made from autofluorescent imaging – in other words, the intrinsic fluorescence from the fixed specimens was used as the basis for the histological contrast seen in the 3D model. One of the models (CS17; approx. 41 days of development) was examined in detail in order to test the resolving power of the OPT technology on these unstained embryos, in relation to structures in the developing nervous system and to determine the feasibility of using the model as a framework for mapping gene expression patterns. Digital OPT sections were compared with corresponding histological sections stained in 3 different ways: standard haematoxylin and eosin stain to visualise cell nuclei, cytoplasm and connective tissue  and immunocytochemistry to detect GAP43 or PAX6 expression. H&E staining distinguishes amongst the ventricular, intermediate and mantle layers in the central nervous system and cranial nerves and ganglia are clearly identifiable. Growth-associated-protein 43 (GAP43) is expressed in growing dendrites and axons  and is expressed in the peripheral nervous system and developing tracts of the central nervous system. A number of genes have been identified, particularly in mouse and chick, that are involved in the specification of different brain regions (e.g. reviewed in  and ). Such gene expression patterns can be used to identify specific brain regions and compare their relative extent in different species (reviewed in  and ). PAX6 expression is well characterized as defining several regions and boundaries in the developing mouse brain [17, 18] and . The effectiveness of using 2D section data to generate 3D expression domains was tested by examining two boundary regions in the CS17 model, the zona limitans intrathalamica (between the dorsal and ventral thalamus) and the midbrain-diencephalon boundary.
Results and Discussion
Many structures within the developing nervous system can be identified in the CS17 OPT model with a minimum defined resolution of approximately 20 μm. The CS17 model also acted as a framework onto which anatomical domains were easily painted and gene expression patterns mapped. OPT models have been successfully generated from CS12 to CS23, and these models will provide a means of analyzing and relating changes in anatomy and gene expression both within individual developmental stages and across developmental time.
In the long-term, our aim is to link the 3D models to an anatomical database and embed both of them within a custom-designed gene expression database in order to create an Electronic Atlas of the Developing Human Brain (http://www.ncl.ac.uk/ihg/EADHB/).
Human embryos were collected from termination of pregnancy material, with appropriate written consent, approval from the Newcastle and North Tyneside NHS Health Authority Joint Ethics Committee and following national guidelines . Embryos were collected into cold PBS, separated from surrounding tissue and fixed overnight in 4% paraformaldehyde at 4°C before short-term storage at 4°C in 70 % ethanol. Placental tissue was sampled for karyotype analysis prior to fixation of the embryo tissue.
Intact specimens were rehydrated through a graded series of ethanol and embedded in a block of 1% low melting point agarose. They were then dehydrated through a graded series of methanol before being cleared using a mixture of benzyl alcohol and benzyl benzoate. 400 digital images were then captured while the now almost transparent specimens were rotated in a full circle, with 0.9° steps between each image. The signal corresponded to the weak autofluorescence originating from the paraformaldehyde-fixed tissue and was detected using a wideband FITC filter with excitation at 465–500 nm and emission from 515–560 nm. The images were then assembled to recreate the 3D shape of the embryo, using modified tomography algorithms .
Post OPT processing and histology
After OPT scanning the CS17 embryo N365 was rehydrated through a graded series of methanol and was then removed from the agarose block by incubation in a 0.29 M sucrose solution at 55°C. It was then processed for paraffin wax embedding and 5 μm microtome sections were cut. Every 5th section was stained with haematoxylin and eosin, following standard procedures.
The remaining N365 sections were used for immunohistochemistry. Alternate one-in-five section series were stained with antibodies against GAP43 (GAP-7B10, Sigma), or PAX6 (PRB-278P, Covance) using standard techniques. The reaction was visualised with diamino benzidine and the sections lightly counterstained with toluidine blue.
Gene Expression Mapping
Images of the stained sections were captured through a ×2.5 objective (as viewed down the microscope at 25× magnification) using the Zeiss Axiovision system. For the PAX6 and GAP43 data, a modified warping interface of the MAPaint software was used to match each stained, physical section to the corresponding digital OPT section. Correspondences between the physical (source) and digital (target) images were identified and manually tie-pointed. The source image was then transformed to the shape of the target section, and the image transformation saved. The interface uses interactive thresholding to extract the expression signal from the source image and then applies the image transformation to map this signal into the space of the 3D OPT model. This was carried out for approximately 190 sections through the head of the CS17 embryo until the full 3D expression pattern was built up for each of the GAP43 and PAX6 data sets.
List of abbreviations
central nervous system
midbrain/ diencephalon boundary
dorsal root ganglion
Electronic Atlas of the Developing Human Brain
Growth Associated Protein 43
haematoxylin and eosin
optical projection tomography
collapsed roof of 4th ventricle
zona limitans intrathalamica
The project is funded by the National Institutes of Health (USA) Human Brain Project. (NIMH and NICHD). The human tissue was provided by the Joint MRC-Wellcome Human Developmental Biology Resource at IHG, Newcastle upon Tyne.
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