Mathematical characterization of three-dimensional gene expression patterns.

MOTIVATION: The importance of a systematic methodology for the mathematical characterization of three-dimensional gene expression patterns in embryonic development. METHODS: By combining lacunarity and multiscale fractal dimension analyses with computer-based methods of three-dimensional reconstruction, it becomes possible to extract new information from in situ hybridization studies. Lacunarity and fractality are appropriate measures for the cloud-like gene activation signals in embryonic tissues. The newly introduced multiscale method provides a natural extension of the fractal dimension concept, being capable of characterizing the fractality of geometrical patterns in terms of spatial scale. This tool can be systematically applied to three-dimensional patterns of gene expression. RESULTS: Applications are illustrated using the three-dimensional expression patterns of the myogenic marker gene Myf5 in a series of differentiating somites of a mouse embryo.

Role of motility in embryonic development I: Embryo movements and amnion contractions in the chick and the influence of illumination.

This study provides a quantitative analysis of the active movements of the chick embryo and of the contractions of the amnion over the entire developmental period of 21 days. Four types of embryo movements are distinguished. The motor activity of the embryo shows two characteristic peaks, with maximum contraction frequencies on the 12th and on the 16th day. In contrast, the amnion activity is higher at earlier stages and decreases as the body activity increases. The amnion activity is largely independent of the body activity. Illumination has a strong influence on embryo movements. It is shown that increases of light intensity affect the patterns of activity of both the embryo and the amnion. While the effect of light on the embryo can be interpreted as being transmitted via the optic system, the mechanism of the amniotic response is unclear. The results suggest that the amnion itself may be sensitive to light. J. Exp. Zool. (Mol. Dev. Evol.) 291:186-194, 2001.

3D modelling of gene expression patterns.

The current genome-sequencing projects provide "word indices" of the book of life. A central post-genomic question will be how these words are three-dimensionally deployed in the generation of organism form. Gene expression studies of developing organisms contribute an increasing wealth of snapshot data on the activation of individual genes at selected locations and single moments in the developmental process. However, a comprehensive understanding of the dynamic activation of multiple genes and their functional role in controlling the 3D processes of collective cell behaviour, pattern formation and morphogenesis, requires special tools for a systematic description of spatio-temporal patterns of gene activation and the ensuing phenotypic effects. This article concentrates on new, computer-based tools for the 3D analysis of gene expression patterns in embryonic development and their use for the systematic establishment of comprehensive gene expression maps.

Computer-based three-dimensional visualization of developmental gene expression.

A broad understanding of the relationship between gene activation, pattern formation and morphogenesis will require adequate tools for three-dimensional and, perhaps four-dimensional, representation and analysis of molecular developmental processes. We present a novel, computer-based method for the 3D visualization of embryonic gene expression and morphological structures from serial sections. The information from these automatically aligned 3D reconstructions exceeds that from single-section and whole-mount visualizations of in situ hybridizations. In addition, these 3D models of gene-expression patterns can become a central component of a future developmental database designed for the collection and presentation of digitized, morphological and gene-expression data. This work is accompanied by a web site (http://www.univie.ac.at/GeneEMAC).

Epigenetic mechanisms of character origination.

The close mapping between genotype and morphological phenotype in many contemporary metazoans has led to the general notion that the evolution of organismal form is a direct consequence of evolving genetic programs. In contrast to this view, we propose that the present relationship between genes and form is a highly derived condition, a product of evolution rather than its precondition. Prior to the biochemical canalization of developmental pathways, and the stabilization of phenotypes, interaction of multicellular organisms with their physicochemical environments dictated a many-to-many mapping between genomes and forms. These forms would have been generated by epigenetic mechanisms: initially physical processes characteristic of condensed, chemically active materials, and later conditional, inductive interactions among the organism's constituent tissues. This concept, that epigenetic mechanisms are the generative agents of morphological character origination, helps to explain findings that are difficult to reconcile with the standard neo-Darwinian model, e.g., the burst of body plans in the early Cambrian, the origins of morphological innovation, homology, and rapid change of form. Our concept entails a new interpretation of the relationship between genes and biological form.

Intimal hyperplasia of the infant parasellar carotid artery: a potential developmental factor in atherosclerosis and SIDS.

Intimal cushions that project into the lumen of arteries are precursors of atherosclerotic plaque formation. The "carotid siphon, " although frequently affected by atherosclerosis, was never analyzed for the occurrence of neonatal intimal hyperplasia. This study provides a topographic and morphometric analysis of intimal cushions in the parasellar internal carotid artery (pICA) of the human infant. A total of 35 specimens were studied in detail, using both standard histological techniques and a new method of computer-aided 3D reconstruction. Intimal hyperplasia occurred at 3 characteristic locations of the pICA: (1) the convex side of the posterior knee (C5 cushion), (2) the bottom of the horizontal segment (C4 cushion), and (3) the concave side of the anterior knee (C3 cushion). The extension of the cushions and the degrees to which they occluded the vessel lumens were measured. The complex shape of the pICA required 3D computer models for exact topographical descriptions and precise measurements. Our results suggest that the occurrence and degree of intimal hyperplasia are related to shape changes of the pICA during postnatal development. We predict that individuals who retain the relatively straight course of the fetal pICA throughout their lives are less prone to develop atherosclerotic lesions at this portion of the carotid artery. A possible contribution of neonatal intimal cushions to the origin of sudden infant death syndrome is discussed.

Generation, integration, autonomy: three steps in the evolution of homology.

The homology concept harbours implicit assumptions about the evolution of morphological organization. Homologues are natural units in the construction of organismal body plans. Their origin and maintenance should represent a key element of a comprehensive theory of morphological evolution. Therefore, it is necessary to understand the causation of homology and to investigate the mechanisms underlying its origination. The study of this issue cannot be limited to the molecular level, because there appears to exist no strict correspondence between genetic and morphological evolution. It is argued that the establishment of homology follows three distinct (if overlapping) steps: (a) the generation of morphological building elements; (b) the integration of new elements into a body plan; and (c) the autonomization of integrated construction units as lineage-specific homologues of phenotypic evolution. In contrast with traditional views, it is proposed that the mechanistic basis for steps (a) and (b) is largely epigenetic, i.e. a consequence of the inherent propensities of developmental system under changing conditions. Step (c) transcends the proximate mechanisms underlying the establishment of homologues and makes them independent attractors of morphological organization at the phenotypic level.

The parasellar region of human infants: cavernous sinus topography and surgical approaches.

OBJECT: In this study the authors analyze the peculiarities of the parasellar anatomy and the topography of surgical approaches to the parasellar region (PSR) in human infants. METHODS: Forty-nine specimens of the PSR obtained at autopsy were studied using microdissection and histological analysis. Important distances between anatomical landmarks were measured with the aid of a dissecting microscope. One serially sectioned specimen was three-dimensionally reconstructed and analyzed on the computer screen by using the authors' new episcopic reconstruction technique. CONCLUSIONS: The anatomy of the infant PSR differs distinctly from that of the adult. The parasellar portion of the internal carotid artery (ICA) does not form a siphon, but takes a straight course, and the venous pathways as well as the cranial and sympathetic nerves have different topographical relationships. Analyses of surgical approaches demonstrate that, in young children, the anterolateral approach can be used to reach the pterygopalatine compartment, the superior ophthalmic vein, and those pathological processes that extend from the orbit into the PSR. The approach via Parkinson's triangle can be used in 45% of cases to access the pathological processes that occur in the voluminous space above and behind the posterior flexure of the parasellar ICA. Taking this route, sympathetic nerve fibers passing through the PSR are not at risk, but some arterial branches that run within the lateral wall of the sinus can complicate this approach. This study presents a guideline that can assist radiologists and neurosurgeons in the planning and performance of interventions within the PSR of neonates and young children.

A new episcopic method for rapid 3-D reconstruction: applications in anatomy and embryology.

The topographic relations of complex structures and the morphogenesis of organ systems can only be fully understood in their three-dimensional context. Three-dimensional (3-D) reconstruction of physically sectioned specimens has become an indispensable tool in modern anatomical and embryological research. Teaching also makes increasingly use of 3-D representations, in particular in the case of embryonic systems that undergo complicated transformations of form and shape. At present no cheap and simple technique is available that generates accurate 3-D models of sectioned objects. In this study we describe a novel technique that rapidly provides faithful 3-D models of sectioned specimens. The images are captured directly from the cutting surface of the embedding block after each sectioning and "on block" staining step. Automatic image processing generates a stack of binary images of the specimen contour. Binary images of internal structures are obtained both by automatic segmentation and manual tracing. Since these image series are inherently aligned, they can be reconstructed three-dimensionally without time-consuming alignment procedures. The quality and the flexibility of the method are demonstrated by reconstructing three kinds of specimens of different histological composition and staining contrast: a 4 mm mouse embryo together with several of its inner organs, a cavernous sinus region of a human infant, and a segment of a human carotid artery. Very short processing times and the faithful representation of complex structural arrangements recommend this technique for routine use in morphological research and for creating embryologic teaching models or 3-D embryonic staging series.

External marker-based automatic congruencing: a new method of 3D reconstruction from serial sections.

BACKGROUND: Computer-based three-dimensional (3D) visualizations reconstructed from sectional images represent a valuable tool in biomedical research and medical diagnosis. Particularly with those imaging techniques that provide virtual sections, such as CT, MRI, and CLSM, 3D reconstructions have become routine. Reconstructions from physical sections, such as those used in histological preparations, have not experienced an equivalent breakthrough, due to inherent shortcomings in sectional preparation that impede automated image-processing and reconstruction. The increased use of molecular techniques in morphological research, however, generates an overwhelming amount of 3D molecular information, stored within series of physical sections. This valuable information can be fully appreciated and interpreted only through an adequate method of 3D visualization. METHODS AND RESULTS: In this paper we present a new method for a reliable and largely automated 3D reconstruction from physically sectioned material. The 'EMAC' concept (External Marker-based Automatic Congruencing) successfully approaches the three major obstacles to automated 3D reconstruction from serial physical sections: misalignment, distortion, and staining variation. It utilizes the objectivity of external markers for realignment of the sectional images and for geometric correction of distortion. A self-adapting dynamic thresholding technique compensates for artifactual staining variation and automatically selects the desired object contours. CONCLUSIONS: Implemented on a low-cost hardware platform, EMAC provides a fast and efficient tool that largely facilitates the use of computer-based 3D visualization for the analysis of complex structural, molecular, and genetic information in morphological research. Due to its conceptual versatility, EMAC can be easily adapted for a broad range of tasks, including all modern molecular-staining techniques, such as immunohistochemistry and in situ hybridization.

Anatomical compartments of the parasellar region: adipose tissue bodies represent intracranial continuations of extracranial spaces.

The cavernous sinus is traditionally described as a single anatomical compartment that contains cranial nerves, blood vessels, and connective tissue. A detailed analysis of 45 infant and 4 fetal parasellar regions shows that this view must be modified. The spatial arrangement, the topographic relations, and the expansion of the adipose and connective tissue spaces were analysed and reconstructed 3-dimensionally on a computer. It is shown that 3 different anatomical compartments, which are strictly demarcated by connective tissue, compose the parasellar region of infants. Two represent intracranial continuations of extracranial tissue spaces. The 3rd compartment corresponds to the so-called 'cavernous sinus' of the adult. Each of the 3 compartments contains characteristic adipose tissue bodies. Because the cavernous sinus represents only one compartment of the area, we propose to use the term 'parasellar region' to designate the entire anatomical region on either side of the sella turcica.

The sympathetic nerves of the parasellar region: pathways to the orbit and the brain.

Sympathetic nerves innervate targets in the orbit and the brain. They issue from the superior cervical ganglion and reach the parasellar region via the internal carotid nerve. Information on their further parasellar course and distribution is scant and contradictory. In this study the parasellar sympathetic pathways of 30 human infants and 6 human fetuses were investigated by microdissection and histologically. A common parasellar sympathetic trunk, which reunites all the nerve fibers emanating from the lateral and medial internal carotid plexus, is described as well as its further divisions. It was found that the posterior knee of the infant carotid siphon is free of large sympathetic nerve bundles. In addition a ganglion is described, which is situated in the parasellar adipose body. It is reached by nerve fibers coming from the parasellar sympathetic pathways. Fibers that issue from this ganglion join the periorbita and the orbital muscle of Müller. These anatomical facts are of immediate importance for preventing nerve damage during cavernous sinus surgery. Furthermore, the study improves the anatomical knowledge of the parasellar region and suggests a new concept for the innervation of the orbital muscle.

A comparative study of stereolithographically modelled skulls of Petralona and Broken Hill: implications for future studies of middle Pleistocene hominid evolution.

Computer generated three-dimensional stereolithographic models of middle Pleistocene skulls from Petralona and Broken Hill are described and compared. The anterior cranial fossae of these models are also compared with that of another middle Pleistocene skull, Arago 21. Stereolithographic modelling reproduces not only the outer surfaces of skulls, but also features within the substance of the bones, and details of the internal braincase. The skulls of Petralona and, to a somewhat lesser degree, Broken Hill are extremely pneumatized. Previously undescribed features associated with pneumatization are detailed, along with their possible functional significance, polarity, and potential for understanding hominid cranial variation. Petralona and Broken Hill also exhibit a dramatic suite of cerebral features that is probably related to extensive pneumatization of the skull, namely frontal lobes that are tilted and located behind rather than over the orbits, laterally flared temporal lobes, marked occipital projection, and basal location of the cerebellum. Comparison of the anterior cranial fossae of Petralona, Broken Hill, and Arago 21 suggests that external resemblance of skulls may not always correlate with endocranial similarity. We believe that stereolithographic reconstructions have the potential for helping to resolve difficult questions about the origins of Neanderthal and anatomically modern people.

[3-dimensional reconstruction of histological serial sections using a computer]. / Dreidimensionale Rekonstruktion histologischer Schnittserien am Computer.

This paper reports the development of an objective and time-saving method for the three-dimensional reconstruction of sectioned morphological specimens on the computer. The development of a fiducial marker technique, the design of program extensions for the automatic thresholding and realignment of images, and a technique for the transfer of marker-derived reference points into images of higher magnification are described. The method is based on the use of resin as embedding material and drill holes as fiducials. Images are digitized by a video camera, and NIH-Image is used for image processing. Special macros were written for the automation of a number of operations, such as object and marker segmentation, image reorientation, image scaling, dirt removal, and marker transfer. The so-treated image series were transferred to a workstation and were reconstructed using "Image Volumes". The reconstructs obtained from this largely automated technique exhibit a perfect alignment of the sections. The 3-D representations of the sectioned objects have a much higher degree of objectivity than those obtained by manual image orientation or by semiautomated techniques that do not use fiducials.

Limb development in a primitive crustacean, Triops longicaudatus: subdivision of the early limb bud gives rise to multibranched limbs.

Recent advances in developmental genetics of Drosophila have uncovered some of the key molecules involved in the positioning and outgrowth of the leg primordia. Although expression patterns of these molecules have been analyzed in several arthropod species, broad comparisons of mechanisms of limb development among arthropods remain somewhat speculative since no detailed studies of limb development exist for crustaceans, the postulated sister group of insects. As a basis for such comparisons, we analysed limb development in a primitive branchiopod crustacean, Triops longicaudatus. Adults have a series of similar limbs with eight branches or lobes that project from the main shaft. Phalloidin staining of developing limbs buds shows the distal epithelial ridge of the early limb bud exhibits eight folds that extend in a dorsal ventral (D/V) arc across the body. These initial folds subsequently form the eight lobes of the adult limb. This study demonstrates that, in a primitive crustacean, branched limbs do not arise via sequential splitting. Current models of limb development based on Drosophila do not provide a mechanism for establishing eight branches along the D/V axis of a segment. Although the events that position limbs on a body segment appear to be conserved between insects and crustaceans, mechanisms of limb branching may not.

Homeotic duplication of the pelvic body segment in regenerating tadpole tails induced by retinoic acid.

Homeosis, the ectopic formation of a body part, is one of the key phenomena that prompted the identification of the essential selector genes controlling body organization. Shared elements of such homeotic genes exist in all studied animal classes, but homeotic transformations of the same order of magnitude as in insects, such as the duplication of the thorax in Drosophila mutants, have not been described in vertebrates. Here we investigate the capacity of retinoic acid to modify tail regeneration in amphibians. We show that retinoic acid causes the formation of an additional body segment in regenerating tails of Rana temporaria tadpoles. A second pelvic section, including vertebral elements, pelvic girdle elements and limb buds, forms at the mid-tail level. This is the first report of a homeotic duplication of a whole body segment in vertebrate axial regeneration.

Ontogeny of the syndesmosis tibiofibularis and the evolution of the bird hindlimb: a caenogenetic feature triggers phenotypic novelty.

The underlying theme of this study is the contribution of developmental mechanisms to the generation of morphological novelty in evolution. The syndesmosis tibiofibularis, an important structural and functional link between the two zeugopod bones of the bird hindlimb, is used as a model for evolutionary novelty. We analyze the structural, developmental and adaptive aspects of its origin in a combined descriptive, experimental, and comparative approach. The ontogeny of the syndesmosis in the chick embryo involves several developmental steps, including the formation of a separate cartilage rudiment that in turn stimulates the formation of an osseous crest on the tibia, which will eventually replace the cartilage element itself. Some of the epigenetic requirements for the formation of the cartilage element and the osseous crest are demonstrated by experimentally increasing the distance between the two zeugopod bones, an operation that results in the absence of both cartilage and crest. Although a syndesmosis tibiofibularis associated with an osseous crest on the tibiotarsus is unique to birds in extant vertebrates, the presence of a distinct crest at the corresponding location in theropod dinosaurs indicates that a syndesmosis also existed in this group of archosaurs. The results of the study suggest that in the case of the syndesmosis tibiofibularis phenotypic evolutionary novelty is based on a caenogenetic feature, i.e. a feature that initially arose in response to changing developmental conditions. In conclusion we propose a model for the stepwise evolutionary modification of the sauropsid hindlimb, integrating adaptive trends and developmental mechanisms that interactively determine the transformations of skeletal limb morphology. The syndesmosis tibiofibularis and the mechanisms of its formation are not only shown to have played a key-role in this process, but its presence in theropod dinosaurs also points towards the origin of birds.