a three-dimensional model of the mouse at embryonic day 9 · developmental biology 216, 457–468...

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Developmental Biology 216, 457–468 (1999)Article ID dbio.1999.9500, available online at http://www.idealibrary.com on

A Three-Dimensional Model of the Mouseat Embryonic Day 9

R. M. Brune,*,1 J. B. L. Bard,* C. Dubreuil,† E. Guest,† W. Hill,†. Kaufman,* M. Stark,† D. Davidson,† and R. A. Baldock†

*Anatomy Section, Biomedical Sciences, University of Edinburgh, Teviot Place, Edinburgh,EH8 9AG, United Kingdom; and †MRC Human Genetics Unit, Western General Hospital,Crewe Road, Edinburgh, EH4 2XU, United Kingdom

This paper describes a digital, three-dimensional model of the mouse embryo at E9. The model was made by reconstructionfrom images of serial histological sections digitally warped to remove distortions and has a resolution of approximately 9mm. The model can be digitally resectioned in any plane to provide images which resemble conventional histologicalsections. The main tissues have been identified and delineated by digital painting so that the anatomical components canbe visualized and manipulated in 3-D surface- and volume-rendered views. This provides a three-dimensional definition ofanatomy that will provide a useful tool for interpreting and understanding spatial data in mouse embryos. The anatomy ofthe model is discussed where it provides landmarks for interpretation and navigation or where it is unexpected in light ofexisting descriptions of the E9 mouse embryo. The complete anatomy is not presented in this paper but will be availableon CD-ROM. A detailed description of the technical aspects of the construction of the model is included in an appendix.The model is the first of a series that will form the basis for an atlas/database of mouse development. This reconstructionand its associated anatomy are available in a variety of data formats with some supporting software fromhttp://genex.hgu.mrc.ac.uk/. © 1999 Academic Press

Key Words: E9 mouse embryo; Theiler stage 14; atlas; anatomy; 3-D reconstruction; voxel images; anatomical
nomenclature.
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INTRODUCTION

The mouse is the major model system for studying thegenetic basis of mammalian development, yet there is nodetailed, systematic reference description of the morpho-logical phenotype of the mouse embryo. Such a descriptionis necessary to help us analyze the vast amount of dataneeded to understand gene function. The description shouldprovide a common framework in which to map and com-pare data on cell activities, gene expression patterns, mu-tant phenotypes, etc. A key aspect of these data is thatmuch of it is essentially spatial and does not necessarilyfollow tissue boundaries; the framework must thereforeaccommodate spatially mapped data as well as informationassigned to named parts of anatomy. In order to permit anunderstanding of tissue dynamics and morphogenesis, sucha framework should combine an overview of the three-

1 To whom correspondence should be addressed. E-mail:[email protected].

0012-1606/99 $30.00Copyright © 1999 by Academic PressAll rights of reproduction in any form reserved.

imensional morphology of organs and tissues with anndication of their histological structure and the distribu-ion of cells. Conventional embryo atlases (Rugh, 1968;heiler, 1989; Kaufman, 1992) can only provide a limitedumber of pictures and section planes. A more completeescription of mouse anatomy that not only details thenatomical structures, but can also be viewed in anyrbitrary orientation or plane of section, is now necessary.To provide such a description, we are creating 3-D digital

oxel2 models of mouse embryos from images of serialhistological sections. We have based the models on conven-tional histological sections, rather than, e.g., MR images, toprovide a view of the material that is familiar to develop-mental biologists. To relate this purely spatial descriptionto anatomy, we have delineated the boundaries of struc-tures that are recognizable with standard staining and that

2 Voxel is the 3-D equivalent of pixel in a 2-D image which is the
picture element representing the image content at that point. Inthis case the voxels are arranged on a rectangular 3-D grid.
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4593-D Model of the E9 Mouse Embryo

are named in the standard anatomy database (Bard et al.,1998). These models will form the framework for one ormore databases in which any spatially organized data, e.g.,gene expression, can be mapped3 (Baldock et al., 1992;Davidson and Baldock, 1997; Davidson et al., 1997). Thesedata can then be analyzed, compared, and interrogated inpurely spatial terms as well as in relation to the underlyinganatomy. The standard anatomical nomenclature, digital

FIG. 3. Three representative sections: (a) through the forebrain, inshown on either side of the remnants of the buccopharyngeal memside of the section). This section is also through the proximal padisplaying the primitive ventricle, bulbus cordis and outflow tractpit is clearly seen. (e) Through the caudal region of the foregut,transversum is seen to be surrounding the hepatic diverticulum; th(Kaufman, 1992, plates 18a and 18b). Toward the bottom right of secis shown, and the same area painted is shown in (b). In the complemof these sections have been displayed using only five colors (it washistology).

3 See http://genex.hgu.mrc.ac.uk/SamplePics/#Gene Expression.

Copyright © 1999 by Academic Press. All right

mbryos, and gene-expression database are being developeds part of the Mouse Gene Expression Information Re-ource4 (MGEIR, Ringwald et al., 1994).In this paper we report the first digital model of the E9 (9

pc, Theiler stage 14) mouse embryo which is now availablen CD-ROM. The model can be digitally resectioned in anyrbitrary plane to show a histological section that corre-ponds to a section through the user’s own material, with

region of the optic vesicles; through the first branchial arches, onein the region of the oral pit; and the hindbrain (shown in the right

the tail (top left of the section). (c) Through the thoracic region,e primitive heart; in the dorsal region of the embryo, the left otic

to the foregut–midgut junction; the rostral part of the septumites, located on either side of the tail region, are also clearly seen

(a), a magnified area of the rostral part of the first pharyngeal pouchary “painted” sections, b, d, and f, the anatomical domains in eachossible to give every tissue its own color and view the underlying

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4 See our WWW site: http://genex.hgu.mrc.ac.uk.

s of reproduction in any form reserved.

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460 Brune et al.

the advantage that all the tissues are delineated and named.Such a model can also be displayed in a volume-renderedformat, the digital equivalent of a whole-mount embryopreparation. This provides a useful tool for relating anyspatial data to anatomy and also as a teaching aid (Kaufmanet al., 1996). We discuss the anatomy of the model where its unexpected in light of existing descriptions of E9 mousenatomy for purposes of presenting the advantages of theystem and aiding the user to navigate through the modelith the help of morphological landmarks.The E9 embryo was chosen for our initial model because

it is intermediate in complexity and therefore provides agood test of our approach and also because it represents animportant period in development at the transition betweenembryogenesis and organogenesis. To ensure consistency ofreference material we have used serial sections from the E9embryo illustrated in the “Atlas of Mouse Development”(Kaufman, 1992) which is typical of this stage.

MATERIAL AND METHODS

Embryos

The E9 embryo used for the model was obtained by mating a(C57BL3CBA)F1 hybrid male with a female of the same geneticconstitution. Preparation of the embryo has already been described(Kaufman, 1992): in summary, the embryo was dissected free of itsextraembryonic membranes (except amnion), fixed in Bouin’s fixa-tive, routinely processed and embedded in wax, and then sectionedat a nominal thickness of 7 mm. Contiguous ribbons of sectionswere collected, ordered, and mounted on glass slides and thenstained with hematoxylin and counterstained with eosin.

Reconstruction

The gray-level (i.e., photographic) voxel model was recon-structed using the full set of 307 histological sections. The require-ment was to produce a 3-D model representative of the E9 mouseembryo that can be used for resectioning at arbitrary orientations toallow mapping of gene-expression data. For this purpose we havedeveloped a technique that produces a reconstruction from a set ofsection images without fiducial marks for registration. The digitalembryo does not, therefore, necessarily have exactly the sameshape as the original specimen but has had the random distortionsarising from the sectioning process largely removed. The overallalignment of the sections was established by demanding that theexternal shape of the final reconstruction be representative of afixed embryo of this stage. The details of the process are given inthe appendix and the result for the E9 embryo is shown in Fig. 1.

Delineating Anatomical Structures

Anatomical structures were delineated using a program(MAPaint) developed for this purpose. This software includes someautomatic functions (e.g., edge detection), but, for the modeldescribed here, we found that much of the delineation had to bedone manually. At any one time, MAPaint allows the user to
delineate up to 32 structures in any arbitrary view (Fig. 2). Theanatomical domains are stored as separate 3-D binary images in the

database and there is no limit, in principle, on how many domainscan be delineated and independently stored.

Our intention has been to delineate the boundaries of all themajor tissues and their constituent parts that can be recognized inthis embryo, not to paint individual cells. Tissues were paintedserially, mostly on images of the original transverse sections, butwith the help of other planes of sectioning where necessary. In allcases, tissue boundaries were established by checking the morphol-ogy of the original sections using a microscope. The delineatedboundaries where subsequently checked and corrected if the errorwas greater than one pixel.

While most intertissue boundaries could be identified throughinspection of the original section, a few had to be determined onthe basis of reconstructed planes (e.g., the somites) or had to bedetermined arbitrarily. In some cases, there was no discontinuity intissue structure in passing from one component to another (e.g.,where the branchial pouches or endodermal linings of the branchialarches meet the anterior foregut); in such cases, the standardapproach was to mark the boundary on the basis of its 3-Dmorphology or its position relative to other tissues (see Figs. 3a–3d).A further problem arose in delineating partially formed structures(e.g., in defining a boundary between the prenotochordal mesen-chyme and the condensed notochord) where the decision must bearbitrary, as there is a developmental transition zone rather than atrue boundary. In other cases, boundaries were difficult to definebecause they were not clearly indicated by the staining (e.g., thatbetween the common atrium and the primitive ventricle). Where aboundary was not clearly defined morphologically on a singlesection, it was placed according to one or more of the following ofcriteria: (i) 3-D morphology, (ii) position of surrounding tissues, (iii)cell arrangement, or (iv) by selecting a boundary half way betweenpositions where the tissue identification is clear. In instanceswhere the placement of a boundary involved informed judgment orarbitrary decision, the basis for placement has been recorded innotes on the CD-ROM.

Visualization

We have used three methods to visualize the gray-level imageand its delineated anatomy:

● Arbitrary digital histological sections through the volume ofthe 3-D model using MAPaint;

● “Whole-mount” 3-D visualization viewed by direct volume-rendering techniques; and

● 3-D visualization of components as surfaces with controllableproperties such as color and transparency (including true stereoviewing with appropriate hardware).

Digital sections. MAPaint (Fig. 2) provides an interface fordisplaying (and modifying) 3-D domains in the embryo model. Theprogram allows the user to view any number of 2-D sectionsthrough the model, each of which can be set at any viewing angle.To assist navigation, it is possible to set either one or two fixed orfiducial points in the model about which the viewed section willrotate. When the appropriate angle has been set, the interfaceprovides a “slider” control to allow the user to pan through the setof parallel sections in that orientation with feedback provided in a3-D navigation window. The required reconstruction is read fromthe Mouse Atlas CD-ROM, and the user is provided with a 3-D
outline view of the embryo and an anatomy “menu” which allowsany of the predefined domains to be read in and visualized. The

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anatomy menu matches the structure of the E9 anatomy hierarchydefined in the online database (Bard et al., 1998).

Within the program, anatomical domains can be shown either asransparent or as solid overlays over the gray-level histology imagend each viewing window provides feedback of the current tissuender the screen pointer. An example of the use of MAPaint toiew structures is shown in Fig. 2. MAPaint was written in C using11/Motif, will run on almost any UNIX-based workstation and isvailable for use with the CD-ROM.

3-D volume rendering. For volume rendering we have used theVisualisation Toolkit” (VTK, Schroeder et al., 1998) to displayiews of the “painted” volume from MAPaint. This allows eachissue to be displayed in a different color to show the positions ofissues in whole-mount preparations. Arbitrary domains definedsing MAPaint can be visualized in this way. Figure 4 shows twoenderings of the embryo: the first is the histology alone and theecond shows a number of tissues in different colors; these could besed, e.g., as landmarks to aid orientation and location.3-D surface rendering. We have used the AVS visualization

ystem (Advanced Visual Systems Inc., U.S.A.) to provide surfaceendered 3-D view of the structure of the anatomical domains. Weave used the AVS modules to convert the 3-D voxel-basedomains to a triangulated surface which can then be viewed in thegeometry viewer.” This allows interactive manipulation of theiewing parameters of each surface including orientation, scale,olor, lighting, reflection properties, transparency, and shading. Forhis work, we used a Silicon Graphics Indigo 2 with 128 MbyteAM and Extreme graphics which allows almost real-time ma-ipulation of the viewed data. This workstation also allows the usef “Crystal Eyes” stereo glasses (StereoGraphics Corporation,.S.A.) in which two views of the data differing by a small viewing

ngle are presented to each eye independently. This results in alear 3-D view of the structures with no loss of colors or otheriewing parameters. Figures 5–7 show some examples of the 3-Dendering that can be provided by the AVS software.

RESULTS

The Model Embryo

The digital embryo can be displayed as if in a 3-Dparaffin block, “trimmed” to demonstrate a selection ofplanes of section that show some of the histology (Fig.1b). Two-dimensional section images in the plane of theoriginal histological sections show the resolution of thehistology and anatomical delineation in the 3-D model.In other, arbitrary, planes of section the resolution isslightly lower since the maximum dimension within apixel is 9 mm (diagonal of a 4 3 4 3 7-mm voxel). Thisresolution is still adequate, however, to discern themorphology and boundaries of most tissues. As for theanatomical resolution, the smallest tissue that was de-lineated is the biliary bud, an approximately globularstructure with a diameter of ;35 mm; the thinnest tissueis the walls of the blood vessels, at only one pixel (4 mm)hick. For some purposes a higher resolution may bemportant; therefore, the original image of the sections,ith a pixel size of 1.36 mm, is provided for reference on

the CD-ROM.Three-dimensional volume rendering gives the appear-


ance of a whole-mount preparation and illustrates the levelof realism achieved by the model (Fig. 4). It is worth notingthat individual tissues and their spatial relationships aremost clearly discerned when the 3-D volume renderedmodel is viewed while rotating (e.g., using AVS). Theexternal appearance of the model embryo is normal andthere seems to be little evidence of shrinkage. There is, forexample, no sign of somites bulging through the surfaceepithelium, an artifact often detected when fixed materialis prepared for SEM. Figure 5a shows a 3-D view of theoutline of the embryo with its amnion removed. Theembryo has almost completed the process of turning: theright-hand somites, dorsal aorta, and umbilical vein and theright side of the mesothelial lining of the intraembryoniccoelom are located more ventrally than those on the left(Figs. 5b and 5c), and the septum transversum (Fig. 7) issituated to the left.

Several other external features confirm that this recon-struction conforms with the standard description of the E9embryo. The first two branchial arches are present, the firsthaving no clear line of demarcation between its maxillaryand mandibular components, the otic placode has indentedto form the otic pit, and the forelimb bud is only justbeginning to form. The cephalic part of the neural tube hasclosed, although the caudal neuropore is still open. There isstill evidence of a primitive streak, but the primitive grooveis no longer present. The degree of curvature of the cranio-caudal axis accords with the standard descriptions for thestage (Theiler, 1972; Kaufman, 1992). The internal organi-zation of this embryo also matches that originally describedand analyzed by Theiler (1972) for this stage: there are 17somites, the nephrogenic cord, nephric duct (Fig. 5c),Rathke’s pouch and the hepatic diverticulum with itsbiliary bud have all formed, and the buccopharyngeal mem-brane has recently ruptured (Fig. 7).

The only apparently unusual feature noted in the modelembryo is a twist in the caudal tip of the future spinal cord.Although this feature seems not to have been described inthe literature, it may not reflect any abnormality, as of 29E9 embryos (same F2 cross) examined, 14 showed thisfeature.

Anatomical domains. Table 1 lists the anatomicalcomponents that were identified in the original histolog-ical sections and delineated in the reconstruction. Itshould be noted that this embryo represents a single timepoint during the interval defined by E9 and thus containsonly a subset of the components listed in the standardnomenclature for the stage as a whole (some form ordisappear during this period). Some tissues named in theanatomical database cannot be resolved or distinguishedat the resolution of the reconstruction and are notindividually represented by painted domains. For ex-ample neural-crest-derived cells cannot be distinguished
morphologically from those derived from mesodermwithout the use of specific markers.

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462 Brune et al.

Anatomical Relationships

Landmarks. The painted anatomical components pro-ide a number of landmarks by which someone with littleraining in vertebrate anatomy can navigate through thembryo in order to locate other features of interest in theodel or in their own specimens of similar stage. For this,

he biologist first finds the plane of section through theeference embryo that best corresponds to that through hisr her own material. This is done by identifying parts ofajor structures cut by the same section in the specimen

FIG. 4. Volume rendered views of the E9 embryo. (a) A whole-mouomites, neural tissue, and gut colored to act as landmarks.IG. 5. 3-D views of the E9 reconstruction with the amnion remo

orange), otic pit (pale orange), somites (dark green), arteries (orangeentricle (magenta), bulbus cordis, and outflow tract (red)] and gut (ord (lime green), head somites (dark yellow), body somites (dark gord (purple), presumptive nephric duct (light lilac), and primitive

nd using these structures in the model to interactivelyefine the corresponding plane. With this plane set, the

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odel can be digitally resectioned, if necessary, slightlydjusting the section planes in different parts of the embryo.omparison of section views with views of whole compo-ents and their 3-D relationships can then provide a frame-ork for detailed examination of the users own sections. A

imilar approach can be used to help interpret whole-mountreparations, matching a volume-rendered view of theodel with the conventional view of the specimen under a

issecting microscope. Using the painted anatomical com-onents as landmarks in volume rendered view or in digital

ew of the E9 reconstruction from histology; (b) as (a) but with heart,

(a) outline of the embryo; (b) neural tissue (yellow), optic vesicles, veins (dark blue), heart [common atrial chamber (lilac), primitiveblue); (c) future brain (yellow), optic vesicles (orange), future spinal, heart (see b), intermediate mesenchyme (dark blue), nephrogenick (light pink) in a translucent white outline of the embryo.

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FIG. 6. Stereo pair view of the vascular system of the E9 embryo set for “parallel gaze.” First arch artery (orange), second arch artery (lightrange), dorsal aorta (yellow), vitelline artery (lemon yellow), umbilical artery (dark green), internal carotid artery (cream), vesselsonnecting internal carotid artery to primary head veins (lime green), primary head veins (dark green), anterior cardinal vein (darkurquoise), common cardinal vein (light blue), posterior cardinal vein (dark blue), umbilical vein (pink), vitelline vein (purple), sinus venosuslavender blue), common atrial chamber (lilac), primitive ventricle (magenta), and outflow tract (red).IG. 7. Gut (transparent blue) with notochord (dark blue), septum transversum (golden yellow), biliary bud (red), mesenchyme of first arch

transparent light green) and second arch (transparent red), branchial pouch of the first arch (solid red) and second arch (purple), lining ofhe first arch (yellow) and second arch (lilac), endodermal component of first branchial membrane (orange), thyroid primordium (green), and
ndodermal component of buccopharyngeal membrane (pink). The inset shows the hepatic diverticulum with the biliary bud without theeptum transversum.
Copyright © 1999 by Academic Press. All rights of reproduction in any form reserved.

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ABLE 1ist of Painted Components in the E9 Embryo

EctodermEndoderm

First arch MesenchymeFirst branchial membrane Ectoderm 1 endoderm

ranchial arch Branchial pouchEctoderm

Second arch EndodermMesenchymeBranchial pouch

Pericardial component CavityMesothelial lining

avities and their linings Intraembryonic coelom Pericardio–peritoneal canal CavityMesothelial lining

Peritoneal component CavityMesothelial lining

ctoderm Surface ectodermRathke’s pouch

MesenchymeHead Facioacoustic neural crest

Trigeminal neural crestSomites 1–5

esenchyme MesenchymeIntermediate mesoderm

Body Lateral mesenchyme SomatopleureSplanchnopleure

Somites 6–18Paraxial mesenchymeSeptum transversum

otochordFirst arch arterySecond arch artery

Cardiovascular system—Arteries Dorsal aorta Left 1 rightInternal carotid artery Left 1 rightUmbilical arteryVitelline artery

Cardiac jellyCommon atrial chamber Endothelium

MyocardiumCardiac jelly

Primitive ventricle Endothelium

Cardiovascular system—HeartMyocardiumCardiac jelly

Outflow tract (includes bulbus cordis) EndotheliumMyocardium

rgan system Sinus venosus Left horn 1 right hornMesentery Dorsal mesocardium

AnteriorCardinal vein Common

Cardiovascular system—Veins PosteriorPrimary head veinsUmbilical vein Left 1 rightVitelline vein Left 1 right

Cardiovascular system—Capillaries Vessels connecting int. car. to head veinsFuture brain

Nervous system Future spinal cordNeural lumen

Sensory organ Ear–inner ear Otic pitEye Optic vesicle

Gut lumenGut Thyroid primordium

Biliary budVisceral organ

Buccopharyngeal membrane Ectoderm 1 endoderm

Urogenital system Nephrogenic chordPresumptive nephric duct

rimitive streakxtraembryonic tissue

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Landmarks that are particularly useful in this context are:

● Rostral tips of foregut and notochord;● Notochord (as a midline landmark);● Tip of Rathke’s pouch;● Caudal tip of optic vesicles;● Edges of otic pit/placode;● Buccopharyngeal membrane (if present);● First branchial membrane;● Rostral tips of primitive ventricle and common atrial

chamber;● Caudal tip of bulbus cordis;● Bulboventricular groove; and● Biliary bud.

The cardiovascular system. Figures 5 and 6 show theeveloping heart and cardiovascular system and illustrateeatures of the anatomy that are hard to discern in sectioned

aterial. For example, Fig. 6 shows that the rostral part ofhe vascular system is more developed than the caudal partnd that the seventh intersegmental arteries (betweenomites) that will form the axial arteries to the forelimb areo more extensive than the others. The posterior cardinalein displays minimal evidence of differentiation comparedo the anterior cardinal vein that is now bifurcating into theessels destined to become the internal and external jugulareins. There is well-defined trabeculation throughout therimitive ventricle and the lesser degree of trabeculationssociated with the wall of the bulbus cordis is consistentith its future role as the precursor of the right ventricle

nd proximal part of the outflow tract.The primitive gut, the branchial arches, and the septum

ransversum. Figure 7 shows the primitive gut with theemnants of the buccopharyngeal membrane, the first andecond branchial arch pouches, the first branchial mem-rane, the thyroid primordium, the septum transversum,nd biliary bud. The rostral tip of the foregut is seen to havesharp point that broadens caudally with dorsal-to-ventralattening. There is one feature that was unexpected anday be an artifact: within the diverticulum, there is a

mall, hollow globular structure some 50 mm in diameterdjacent to the bud (Figs. 3e and 3f).Neural tissue. Figure 5c shows a view of the neural tube

n combination with the somites, intermediate mesen-hyme, optic vesicles, and the heart, all within translucenturface epithelium. The optic vesicles point in a posterosu-erolateral direction (like “horns”) and five rhombomeresre visible. It should also be noted that there is no clearxternal morphological demarcation between the caudalart of the future hindbrain and the most rostral part of theuture spinal cord. For this model we define this borderetween the fourth and the fifth somite as shown in Fig. 5c.n addition, the facioacoustic and trigeminal neural-crest-erived preganglionic condensations are first clearly seen athis stage (Figs. 3a and 3b). The notochord has a sharp
ostral boundary dorsal to Rathke’s pouch, but its caudaloundary is ill defined as its tissue merges into the primi-

ive streak at the caudal part of the embryo (Figs. 5c, 7a, andb).Urogenital tissue. The partitioning of intermediate me-

oderm into the nephric cord (kidney precursor tissue) andhe nephric duct has only just started at this stage. Figure 5chows these tissues, together with the somites within theranslucent ectoderm. There is asymmetry between the leftnd right sides. On the left side, the subdivision of thentermediate mesoderm into nephrogenic tissue (medially)nd pronephric duct (laterally) occurs at the level of somite3. On the right side, however, it occurs almost oneomite’s length more caudally, at the level of somite 14. Athe caudal end of the embryo, however, there are noifferences between the axial levels of the right and leftudiments and these levels coincide with the caudal level ofomite 17.Size. One additional set of data that can readily be

xtracted from the reconstruction is the relative volumes ofhe different anatomical tissues (Table 2). Even allowing forny fixation artifacts in this single embryo, some clearbservations can be made. Although the largest singleifferentiated tissue is clearly the neural tissue (19%), it isbvious that a large volume of the embryo unambiguouslyerives from the mesoderm (53%), some of which haslearly started to differentiate, e.g., intermediate meso-erm, somites, heart, and vascular system. Nevertheless,8% of the mesenchyme at this stage still seem undiffer-ntiated, although its fate may already have been deter-ined. What is perhaps surprising is how little of the tissue

an be seen as being derived from endoderm: the gut, itsargest derivative by far, occupies less than 3% of the tissue.

DISCUSSION

The reconstruction described here shows that it is pos-sible to make a detailed 3-D digital model of the Theilerstage 14 embryo and indicates how it can be used toelucidate information about anatomy not readily obtainableusing conventional approaches. It provides an accessiblereference description of the E9 embryo, to which develop-mental data of any sort can be related and in which both thehistological and the anatomical structure can be displayedin a view that matches the individual investigator’s mate-rial. The model can assist structure identification in theembryo; indeed, the delineated anatomical domains in themodel provide a definition of the terms used in the ana-tomical nomenclature database.

The model embryo uniquely provides a view of thestained histological structure of a single embryo that can beexplored in three dimensions and so provides a powerfultool to investigate morphogenesis. The ability to demon-strate discrete anatomical domains in isolation, in combi-nation with other tissues, or within a transparent image ofthe entire embryo can greatly assist us in understanding
various relationships between anatomical components. Forexample, the relationship between the somites and the

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466 Brune et al.

nephrogenic cords and their ducts can now be seen and fullyappreciated for the first time and provide useful landmarks.

In addition to the general features of the anatomy de-scribed in this paper, detailed study of the model may revealfurther features that have not been detected in unstainedwhole embryos and have not been apparent from studies ofserial, stained sections viewed individually. The combineduse of this model with those of younger and older embryoscan be expected to provide novel insight into morphoge-netic processes and the changing spatial relationships be-tween structures during mouse development. In addition,and within the limitations discussed below, the modelprovides a reference against which to analyze the pheno-types of, for example, mutant embryos at the appropriatestage of development.

This reconstruction incorporates all the major anatomi-cal features for its stage of development, but if any tissueneeds to be further subdivided, or changed, this can be

TABLE 2Tissue Volumes (Total Volume of Embryo Approximately 0.5 mm

Tissue name

Tissues derived from ectodermTissues derived from mesodermTissues derived from endodermBody cavities

Gut lumenIntraembryonic coelomNeural lumenOtic lumen

Heart (incl. lumen)Blood vessels

VenousArterial

Central nervous systemFuture brainFuture spinal cordCentral nervous system plus optic vesicles

Urogenital systemFirst branchial arch (incl. artery 1 blood)Second branchial arch (incl. artery 1 blood)Total gutMesenchyme

HeadHead somitesFirst branchial arch (incl. artery 1 lumen)Second branchial arch (incl. artery 1 lumen)BodyBody somitesIntermediateLateral plateParaxialSeptum transversum

readily incorporated into the model. Once the graphicalgene-expression database is established and key gene-


xpression patterns are mapped, we anticipate that thelassical morphological anatomy in the present version ofhe model will be complemented by a molecular anatomy.his will not only help us to perceive the development ofnderlying mechanisms, but will also define new landmarkomains in the embryo.There is one further advantage to this reconstruction that

s worth mentioning. As the various anatomical compo-ents of this typical E9 mouse embryo are very similar tohose present in the Carnegie stage 11 human embryo, it isossible, with little difficulty, to use this mouse reconstruc-ion to help study serial sections of human embryos at thistage (usefulness for navigation is limited to the earlytages).

Limitations of the Model

The most important limitation of the model is that it is

Tissue volume(% whole embryo)

Tissue volume(% of larger component)

25522.8

181550341

7.48.6

3961

186832

190.24.21.02.8

31442.09.42.23.9

122.2

12102.3

3)

based on a single embryo. It can thus only represent onemoment during the interval defined by E9 and gives no


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indication of the variation in morphology, or any hetero-chrony, in the development of different organ systems thatmight be found in a population of the same strain orbetween different strains. The model and the associateddatabase can however be used as a baseline description forsuch data.

Similarly, care must be exercised in attempting to use themodel in any detailed morphometric analysis, for example,as a standard against which to detect subtle changes ofshape in an embryo carrying a mutation. It would bedifficult to justify the use of any standard model for such ananalysis since artifacts arising from differences in fixationand histological processing confound the comparison. Forsuch a purpose, comparisons would normally be madebetween numbers of wild-type and mutant embryos pro-cessed in parallel. Once these differences have been deter-mined by such a method, the results could, however, berecorded in the present model, for example, by delineatingthe normal structures affected by the mutant phenotypeand, perhaps, attaching original images of the mutantphenotype or other data to the appropriate domain. Thisapproach would permit the construction of a searchabledatabase of mutant phenotype and allow them to be com-pared with gene-expression patterns similarly mapped ontothe reference model.

Our choice of specimen also dictates, in part, the resolu-tion of the model, determined by the thickness of thehistological sections. For models of early embryos, duringgastrulation and the formation of the first few somites, wehave used material processed specifically for this work,with a nominal section thickness of 2 mm, which allows aesolution that distinguishes most individual cells in thembryo. The use of an equivalent resolution for the E9mbryo would require a much larger image which wouldeverely reduce the potential for interactive viewing. In theuture, models of selected organs (special systems) at higheresolution may be constructed as necessary and embeddedithin the framework of the whole embryo model. For theresent, we intend to make available images of the fulleries of the original histological sections on which theeconstruction was based. These images will have a pixelize 1.36 3 1.36 mm and will thus provide a higher resolu-

tion at any position in the model.

CONCLUSION

We are making this detailed description widely availableand hope that those with an interest in particular develop-ing systems can begin to incorporate this information intotheir research and, perhaps, comment to us on its uses andlimitations. Where appropriate, information (attributed)may be added to the model so that any investigator canretrieve it.

This model is part of a series, which will form the
framework for a WWW-based, graphical gene-expressiondatabase (Davidson et al., 1997). At present, however,

esearch workers using UNIX workstations can use theodel as an aid to investigating the deployment of three-

imensional gene expression in the E9 embryo using therototype MAPaint program. This can be used immediatelyor 3-D analysis and for storing simple, or partial, gene-xpression patterns in preparation for later entry into theatabase. Details of how to obtain a copy of the model andAPaint can be found on the Mouse Atlas WWW site,

ttp://genex.hgu.mrc.ac.uk/. This site also details the cur-ent status of WWW-based software for accessing, mapping,nd analyzing gene-expression patterns and the graphicalene-expression database.

APPENDIX

Voxel Model Reconstruction

The key requirement of the reconstruction process is toproduce a voxel model that is representative of the E9mouse embryo that can be used for resectioning and withstructures visible using standard histological staining. Ad-ditionally we want the model to be close to the existing defacto standard (Kaufman, 1992). We therefore have devel-oped a reconstruction technique that allows the model to bebuilt from the full set of 307 serial sections, some of whichwere used in the paper atlas. The parts of the process weredigitization, image alignment, image warping, and restack-ing, to form a 3-D gray-level digital model. All stages of thisprocess use proprietary software and hardware systemsdeveloped at the MRC Human Genetics Unit and based onthe woolz image processing system (Piper and Rutovitz,1986).

Image Capture and Alignment

A digital image of each section was recorded using a ZeissAxioplan microscope fitted with a 53 Neofluor objectivelens and a Xillix 1400 (Optimum Vision Ltd., Petersfield,Hampshire, UK) digital camera linked to a Sun Microsys-tems Sparc 10. The Xillix camera provides a digital imagewith a 12-bit gray value (values 0–4095) for each pixelwhich was converted to 8 bit (values 0–255) and then shadecorrected in the usual way (Baldock and Poole, 1993) toremove the effects of uneven illumination and camerasensitivity.

The sections used from the “Atlas of Mouse Develop-ment” (Kaufman, 1992) were cut without any fiducialmarkers; therefore, during capture, the image of each his-tological section was aligned by using a transparent overlayof the preceding section image in the series. This processproduced a stack of registered images ready for the processof warping, which corrects any small registration errors aswell as distortions arising from the sectioning process. Thestack of images was reviewed using the MRC “Recon-
struct” software to correct relative registration errors and toreplace bad sections with adjacent ones. The overall 3-D

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468 Brune et al.

shape of the embryo was checked by viewing the coronaland sagittal sections in MAPaint and adjusting as required.

Warping

Sectioning and histological processing introduce bothsystematic and random distortions to each section whichmust be corrected if arbitrary sectioning through the modelembryo is to generate a realistic image of a section. A keydevelopment for this project has been the production ofwarping software to correct such random distortions(Guest, 1994; Guest and Baldock, 1996) by modeling eachoriginal section image as a thin elastic plate. The basicassumption is that the biological structures visible withinthe gray-level images are smooth on the scale of the sectionthickness. In digital image terms, this implies that, if agiven point on a tissue section is connected by a straightline in 3-D to the corresponding point in the section aboveand similarly to the section below, then the two lines willbe nearly parallel, i.e., there are no sharp corners. Eachsection is modeled as a thin elastic plate and linked to thetwo adjacent sections by “springs” defined by an automaticmatching process. The springs deform the sections tosmooth out tissue boundaries and the elastic properties ofeach plate resist excessive deformation so that the originalembryo shape is retained. The entire process is fully auto-matic.

Subsampling

The original 2-D images of successive sections are com-posed of 1.36 3 1.36 mm pixels the depth of the voxels in theolume image is the thickness of the sections, i.e., 7 mm. To

minimize interpolation when resectioning at an arbitraryorientation, the voxel array should be isotropic. On a cubiclattice the best option would be to have isodiametric voxels,i.e., 7 3 7 3 7 mm. We have compromised by resizing to 4 3

3 7 mm: this allowed the retention of adequate resolutionithout significantly affecting the appearance of the resec-

ioned model. For visualization, the voxels are scaled to give aniform magnification (although the underlying resolutionannot of course be changed). After warping, Gaussian sub-ampling (Gaussian smoothing followed by subsampling:oenderink, 1984) was used to reduce the resolution by a

actor of three in each direction in the original planes.

ACKNOWLEDGMENTS

The authors thank Professor Peter Holland and Dr. David
Wilkinson for helpful discussions on the head–body division.BBSRC and MRC grants funded part of this work.

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Kaufman, M. H. (1992). “The Atlas of Mouse Development.”Academic Press, London.

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Ringwald, M., Baldock, R. A., Bard, J. B. L., Kaufman, M. H., Eppig,J. T., Richardson, J. E., Nadeau, J. H., and Davidson, D. R. (1994).A database for mouse development. Science 265, 2033–2034.

Rugh, R. (1968) “The Mouse: Its Reproduction and Development.”Burgess, Minneapolis. [reprinted 1990, Oxford Univ. Press, Ox-ford]

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Received for publication March 10, 1999
Revised September 8, 1999
Accepted September 8, 1999


a three-dimensional model of the mouse at embryonic day 9 · developmental biology 216, 457–468...

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