BACKGROUND
brain development, particularly in human, involves complex changes in shape and structure over time.
during a period of approximately  <dig> weeks  the major subregions of the human brain are established and development proceeds from a simple neuroepithelial tube to a highly complex three dimensional  structure  <cit> .
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  or, more recently, on computer-based methods, although these are still labour intensive  <cit> .
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  <cit> .
recently, a new, rapid and non-invasive 3d modelling method, optical projection tomography , 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 cs <dig>  <cit> .
mri is likely to be useful for specimens larger than cs <dig> 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 .
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  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  <dig> different ways: standard haematoxylin and eosin stain to visualise cell nuclei, cytoplasm and connective tissue  <cit>  and immunocytochemistry to detect gap <dig> or pax <dig> 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  <dig>  is expressed in growing dendrites and axons  <cit>  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 .
such gene expression patterns can be used to identify specific brain regions and compare their relative extent in different species .
pax <dig> expression is well characterized as defining several regions and boundaries in the developing mouse brain  <cit>  and  <cit> .
the effectiveness of using 2d section data to generate 3d expression domains was tested by examining two boundary regions in the cs <dig> model, the zona limitans intrathalamica  and the midbrain-diencephalon boundary.
RESULTS
an opt model was generated from an intact, unstained embryo which had been staged according to the carnegie staging protocol  <cit>  modified for use with individual embryos rather than in comparisons of many embryos simultaneously  <cit> .
the embryo was staged as cs <dig>  which is approximately  <dig> days of development.
the developing central nervous system  is clearly visible even in the external view of the cs <dig> model .
differences in autofluorescence within the cns and among different organs are also apparent.
there is very little detail in the developing liver, for example, compared to the cns.
blood vessels, dorsal root ganglia and developing vertebrae are clearly visible.
following opt the embryo was embedded in paraffin wax and sectioned using standard methods and then every fifth section was either stained with haematoxylin and eosin or immunocytochemically with antibodies against gap <dig> or pax <dig>  the actual plane of sectioning was identified in the opt model by manipulating the model in mapaint.
this permitted a matched series of digital and physical sections to be compared  in order to assess the resolution of internal structures within the cs <dig> opt model.
different features are highlighted by the different staining techniques and some examples of these are also shown in figure  <dig>  the cranial nerve ganglia stain partially with gap <dig>  and in the h&e sections  and are clearly visible in the opt model .
cranial nerves also stain for gap <dig>  but are less visible in both the h&e section and the opt model.
surprisingly the rootlets of cranial nerve xii do show up clearly in the opt model .
in the cns, the ventricular layer can be clearly distinguished as more darkly staining in the h&e sections and it is also visible as a darker layer in the opt model .
the core of the zona limitans intrathalamica  is seen as a pale region in all three sections .
the gap <dig> stained fibres in the floor of the hindbrain  show clearly as a pale area in the opt model .
as was visible in figure  <dig>  blood vessels and dorsal root ganglia show up very clearly in the opt model .
fluid filled spaces are also clearly visible in the opt model, such as the diencephalic, midbrain and hindbrain ventricles and the otic vesicle .
some artefactual changes in shape seen in the physical sections  are not present in the opt model .
the size of a variety of structures was measured in the h&e and/or gap <dig> stained sections in order to determine the resolution in the cs <dig> model.
when opt is performed on specimens in which specific cells have been fluorescently-labelled, then these small structures can be detected .
however, in cases where the specimen contains no tissue-specific dyes, a resolution of  <dig> to  <dig> micron has previously been found  <cit> .
at the magnification used to generate this cs <dig> model, the smallest clearly measurable structures are the rootlets of the xii cranial nerve which are approximately  <dig> μm in diameter .
mapaint, a unix-based software suite, allows the opt models to be digitally sectioned in any plane and several planes to be viewed simultaneously.
this is illustrated for the cs <dig> model in figure 3a  which moves through a series of digital transverse sections with the corresponding position of each section shown on a sagittal section, followed by a series of sagittal sections and the corresponding position on a transverse section.
the cs <dig> model is shown with some painted anatomical domains and figure 3b shows a snapshot of all fourteen 3d domains with the position of the two transverse sections shown in figure 3c indicated in white.
the anatomical domains were painted in one plane and then checked and refined by corrections introduced in orthogonal planes.
the ease of manipulation of the model means that the best digital plane  can be selected for each anatomical region being painted.
the authors who did these tracings actually found the experience intellectually rewarding, since it amounted to being able to check instantly any difficult point upon a collection of identical specimens sectioned in many different planes.
doubts that often remain unresolved could be resolved very convincingly.
as described in the methods section, and in the legend to figure  <dig>  paraffin sections were immunostained with either anti-gap <dig> or anti-pax <dig> antibodies at approximately  <dig> μm intervals throughout the head of the cs <dig> specimen and the data were captured and mapped to the cs <dig> model.
figure 4a illustrates a sagittal section through the model with digital gap <dig> expression  which has been generated from the data thresholded from transverse sections, an example of which is shown in figure 4b.
there is an unexpected apparent region of no gap <dig> expression in the hindbrain on the sagittal section .
however, the ability to relate the sagittal and transverse sections in the model makes clear that the lack of expression is due to the sagittal section being "cut" obliquely and the section is passing through the floor plate region of the hindbrain at that point, where there is no gap <dig> expression .
a sagittal section with digital pax <dig> expression shown in green also illustrates the lack of pax <dig> expression in the same floor plate region in the hindbrain .
comparing figure 4a and 4c it can be seen that the boundary of the pax <dig> expression in the caudal diencephalon is approximately the same boundary as that of gap <dig> which stains the posterior commissure, just caudal to which is the boundary between the pretectum, the most caudal region of the developing diencephalon  <cit>  and the midbrain.
the lack of pax <dig> and gap <dig> expression in the region just caudal to the posterior commissure is shown  while the expression of both gap <dig> and pax <dig> in the pretectum is shown in the lower two panels of figure 4d.
the 3d pax <dig> domain identifies both the diencephalon-midbrain boundary and the negative intrathalamic boundary or zli .
additional limits of pax <dig> expression visible in figure 5b correspond to the basal telencephalon  and the alar-basal boundary across the entire diencephalon  <cit> .
separate areas of pax <dig> expression appear in the hindbrain
the period from cs <dig>  to cs <dig>  is important because during this time all the major regions of the developing brain are established  <cit>  and most major congenital abnormalities can arise.
major changes take place in the size, shape and complexity of the developing brain during this time and we tested the feasibility of generating opt models throughout this period.
there is a more than  <dig> fold increase in volume between the cs <dig> embryo and the cs <dig> head, however, opt models have been generated successfully throughout these stages.
figure  <dig> shows snapshots of models at each of the stages from cs <dig> to cs <dig>  these are not to scale because each model is generated at the maximum magnification possible, which varies according to the size of the specimen.
the changes in shape and complexity are clearly seen even in these static images.
we currently have  <dig> opt models, including a male and female at each stage cs12-cs <dig> and cs <dig>  at cs <dig> we have twelve different opt models and have assessed their natural variability for  <dig> specific neurodevelopmental features .
at this stage, there was little variation in the features assessed .
movies of all these models can be viewed at our website , which includes a database of gene expression.
the models are too large to display fully or to manipulate online, but a selection of the full models are available on cd on request and a new viewer, the java atlas viewer, has been developed which enables the models to be viewed and manipulated on many platforms .
see additional files  <dig> and  <dig> for the request form and academic use licence agreement.
CONCLUSIONS
many structures within the developing nervous system can be identified in the cs <dig> opt model with a minimum defined resolution of approximately  <dig> μm.
the cs <dig> 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 cs <dig> to cs <dig>  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 .

 brain development, particularly in human, involves complex changes in shape and structure over time. during a period of approximately weeks the major subregions of the human brain are established and development proceeds from a simple neuroepithelial tube to a highly complex three dimensional 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 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, 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 cs. mri is likely to be useful for specimens larger than cs 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. 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 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 different ways: standard haematoxylin and eosin stain to visualise cell nuclei, cytoplasm and connective tissue and immunocytochemistry to detect gap or pax 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 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. such gene expression patterns can be used to identify specific brain regions and compare their relative extent in different species. pax expression is well characterized as defining several regions and boundaries in the developing mouse brain and. the effectiveness of using 2d section data to generate 3d expression domains was tested by examining two boundary regions in the cs model, the zona limitans intrathalamica and the midbrain-diencephalon boundary.  an opt model was generated from an intact, unstained embryo which had been staged according to the carnegie staging protocol modified for use with individual embryos rather than in comparisons of many embryos simultaneously. the embryo was staged as cs which is approximately days of development. the developing central nervous system is clearly visible even in the external view of the cs model. differences in autofluorescence within the cns and among different organs are also apparent. there is very little detail in the developing liver, for example, compared to the cns. blood vessels, dorsal root ganglia and developing vertebrae are clearly visible. following opt the embryo was embedded in paraffin wax and sectioned using standard methods and then every fifth section was either stained with haematoxylin and eosin or immunocytochemically with antibodies against gap or pax the actual plane of sectioning was identified in the opt model by manipulating the model in mapaint. this permitted a matched series of digital and physical sections to be compared in order to assess the resolution of internal structures within the cs opt model. different features are highlighted by the different staining techniques and some examples of these are also shown in figure the cranial nerve ganglia stain partially with gap and in the h&e sections and are clearly visible in the opt model. cranial nerves also stain for gap but are less visible in both the h&e section and the opt model. surprisingly the rootlets of cranial nerve xii do show up clearly in the opt model. in the cns, the ventricular layer can be clearly distinguished as more darkly staining in the h&e sections and it is also visible as a darker layer in the opt model. the core of the zona limitans intrathalamica is seen as a pale region in all three sections. the gap stained fibres in the floor of the hindbrain show clearly as a pale area in the opt model. as was visible in figure blood vessels and dorsal root ganglia show up very clearly in the opt model. fluid filled spaces are also clearly visible in the opt model, such as the diencephalic, midbrain and hindbrain ventricles and the otic vesicle. some artefactual changes in shape seen in the physical sections are not present in the opt model. the size of a variety of structures was measured in the h&e and/or gap stained sections in order to determine the resolution in the cs model. when opt is performed on specimens in which specific cells have been fluorescently-labelled, then these small structures can be detected. however, in cases where the specimen contains no tissue-specific dyes, a resolution of to micron has previously been found. at the magnification used to generate this cs model, the smallest clearly measurable structures are the rootlets of the xii cranial nerve which are approximately μm in diameter. mapaint, a unix-based software suite, allows the opt models to be digitally sectioned in any plane and several planes to be viewed simultaneously. this is illustrated for the cs model in figure 3a which moves through a series of digital transverse sections with the corresponding position of each section shown on a sagittal section, followed by a series of sagittal sections and the corresponding position on a transverse section. the cs model is shown with some painted anatomical domains and figure 3b shows a snapshot of all fourteen 3d domains with the position of the two transverse sections shown in figure 3c indicated in white. the anatomical domains were painted in one plane and then checked and refined by corrections introduced in orthogonal planes. the ease of manipulation of the model means that the best digital plane can be selected for each anatomical region being painted. the authors who did these tracings actually found the experience intellectually rewarding, since it amounted to being able to check instantly any difficult point upon a collection of identical specimens sectioned in many different planes. doubts that often remain unresolved could be resolved very convincingly. as described in the methods section, and in the legend to figure paraffin sections were immunostained with either anti-gap or anti-pax antibodies at approximately μm intervals throughout the head of the cs specimen and the data were captured and mapped to the cs model. figure 4a illustrates a sagittal section through the model with digital gap expression which has been generated from the data thresholded from transverse sections, an example of which is shown in figure 4b. there is an unexpected apparent region of no gap expression in the hindbrain on the sagittal section. however, the ability to relate the sagittal and transverse sections in the model makes clear that the lack of expression is due to the sagittal section being "cut" obliquely and the section is passing through the floor plate region of the hindbrain at that point, where there is no gap expression. a sagittal section with digital pax expression shown in green also illustrates the lack of pax expression in the same floor plate region in the hindbrain. comparing figure 4a and 4c it can be seen that the boundary of the pax expression in the caudal diencephalon is approximately the same boundary as that of gap which stains the posterior commissure, just caudal to which is the boundary between the pretectum, the most caudal region of the developing diencephalon and the midbrain. the lack of pax and gap expression in the region just caudal to the posterior commissure is shown while the expression of both gap and pax in the pretectum is shown in the lower two panels of figure 4d. the 3d pax domain identifies both the diencephalon-midbrain boundary and the negative intrathalamic boundary or zli. additional limits of pax expression visible in figure 5b correspond to the basal telencephalon and the alar-basal boundary across the entire diencephalon. separate areas of pax expression appear in the hindbrain the period from cs to cs is important because during this time all the major regions of the developing brain are established and most major congenital abnormalities can arise. major changes take place in the size, shape and complexity of the developing brain during this time and we tested the feasibility of generating opt models throughout this period. there is a more than fold increase in volume between the cs embryo and the cs head, however, opt models have been generated successfully throughout these stages. figure shows snapshots of models at each of the stages from cs to cs these are not to scale because each model is generated at the maximum magnification possible, which varies according to the size of the specimen. the changes in shape and complexity are clearly seen even in these static images. we currently have opt models, including a male and female at each stage cs12-cs and cs at cs we have twelve different opt models and have assessed their natural variability for specific neurodevelopmental features. at this stage, there was little variation in the features assessed. movies of all these models can be viewed at our website, which includes a database of gene expression. the models are too large to display fully or to manipulate online, but a selection of the full models are available on cd on request and a new viewer, the java atlas viewer, has been developed which enables the models to be viewed and manipulated on many platforms. see additional files and for the request form and academic use licence agreement.  many structures within the developing nervous system can be identified in the cs opt model with a minimum defined resolution of approximately μm. the cs 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 cs to cs 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. 
optical projection tomography is a new technology which has been developed for rapidly generating digital 3d models of intact specimens. despite the 3d models being of unstained tissue, peripheral nervous system structures from the trigeminal ganglion to the rootlets of cranial nerve xii were clearly identifiable, as were structures in the developing neural tube such as the zona limitans intrathalamica. resolution of the opt models was assessed by comparison of digital sections with physical sections stained, either with haematoxylin and eosin or by immunocytochemistry for gap or pax to identify specific anatomical features. 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. the feasibility of applying the opt technology to all stages from cs to cs which encompasses the major period of organogenesis for the human developing central nervous system, was successfully demonstrated. as development proceeds the human embryo attains an ever more complex three dimensional 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. fourteen anatomical domains have been identified and visualised within the cs model. in the cs model considerable detail is visible within the developing nervous system at a minimum resolution of ~ μm and 3d anatomical and gene expression domains can be defined and visualised successfully. we have assessed the resolution of unstained neuronal structures within a carnegie stage opt model and tested its use as a framework onto which anatomical structures can be defined and gene expression data mapped. two 3d gene expression domains, known to be defined by pax expression in the mouse, were clearly visible when pax data from 2d sections were mapped to the cs model.
 as development proceeds the human embryo attains an ever more complex three dimensional 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 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 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 or by immunocytochemistry for gap or pax to identify specific anatomical features. despite the 3d models being of unstained tissue, peripheral nervous system structures from the trigeminal ganglion to the rootlets of cranial nerve xii were clearly identifiable, as were structures in the developing neural tube such as the zona limitans intrathalamica. fourteen anatomical domains have been identified and visualised within the cs model. two 3d gene expression domains, known to be defined by pax expression in the mouse, were clearly visible when pax data from 2d sections were mapped to the cs model. the feasibility of applying the opt technology to all stages from cs to cs which encompasses the major period of organogenesis for the human developing central nervous system, was successfully demonstrated.  in the cs model considerable detail is visible within the developing nervous system at a minimum resolution of ~ 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. 