Johannes Streicher

External marker‐based automatic congruencing: A new method of 3D reconstruction from serial sections

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.

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).

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

SummaryThe 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 with 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.

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

Abstract 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.

Toward a New Reference Method for the Leukocyte Five-Part Differential

A flow cytometric method performing a five-part leukocyte differential based on three-color staining with anti-CD45-fluorescein isothiocyanate (FITC), anti-CD-14-phycoerythrin (PE)/Cy5, and a cocktail of PE-labeled anti-CD2, anti-CD16, and anti-HLA-DR antibodies was evaluated. Results obtained by using three different sample preparation procedures and two different flow cytometers were compared with those of a 1,000-cell manual differential for evaluation of accuracy. We observed excellent correlations with the manual differential for all leukocyte subclasses and even higher correlations between the different flow cytometric methods. Flow cytometric basophil results were identical to the manual counts, regardless of which sample preparation technique or flow cytometer was used. Therefore, we propose our flow cytometric method as the first acceptable automated reference method for basophil counting. The flow cytometric results for the other leukocyte subclasses were apparently influenced by the sample preparation, which could not be explained by cell loss during washing steps. Moreover, a small influence of the flow cytometer was also observed. Assessing the influence of sample storage, we found only minimal changes within 24 h. In establishing reference values, high precision of flow cytometric results facilitated detection of a significantly higher monocyte count for males (relative count: 7.08 +/- 1.73% vs. 6.44 +/- 1.33%, P < 0.05; absolute count: 0.536 +/- 0.181 x 10(9)/liter vs. 0.456 +/- 139 x 10(9)/liter, P < 0.01). Our data indicate that monoclonal antibody-based flow cytometry is a highly suitable reference method for the five-part differential: It also shows, however, that studies will have to put more emphasis on methodological issues to define a method that shows a high interlaboratory reproducibility.

Muscle spindles and Golgi tendon organs in bovine calf extraocular muscle studied by means of double-fluorescent labeling, electron microscopy, and three-dimensional reconstruction

In the present study muscle spindles (MSps) and Golgi tendon organs (GTOs) in bovine extraocular muscles (EOMs) were analyzed in detail. The innervation pattern of these proprioceptors was investigated with transmission electron microscope and confocal laser scanning microscope after double-fluorescent labeling. Three-dimensional (3D) reconstructions were performed of GTOs. Muscle spindles. MSps are numerous, each containing two nuclear bag fibers and up to eight nuclear chain fibers. In the equatorial region and paraequatorial region thin axons enwrapping the intrafusal muscle fibers form numerous nerve contacts on the muscle fiber surface. Double staining of such nerve terminals with synaptophysin and alpha-bungarotoxin and their fine structural features confirm their sensory nature. In the encapsulated part of the polar region neuromuscular contacts have structural features of motor nerve terminals and stain positively with alpha-bungarotoxin. Golgi tendon organs. GTOs are numerous in bovine EOMs. Each GTO contains collagen bundles but frequently also intracapsular muscle fibers. Intracapsular muscle fibers either terminate inside the GTO in collagen bundles or pass through the proprioceptor. GTOs are richly supplied with sensory nerve terminals which intermingle with the collagen bundles. Nerve terminals on intracapsular muscle fibers exhibit fine structural characteristics of motor nerve terminals and are alpha-bungarotoxin positive. The 3D images of GTOs show the detailed spatial arrangement of the GTO tissue components. These new insights in the complex and specific morphology of MSps and GTOs in bovine EOMs indicate that we deal with highly developed proprioceptors. These are supposed to provide important information for EOM innervation.

Natural and experimental reduction of the avian fibula: Developmental thresholds and evolutionary constraint

Fibula reduction is a key feature of avian limb evolution. In a combined comparative and experimental approach the present study analyses the trends of fibula reduction in extant birds and their developmental basis. The study of 55 species of birds reveals four different types of tibiotarsus‐to‐fibula relationships. Extremely small fibulae are associated with two types of limb modification: (1) elongations of the limb primarily affect the tibiotarsus, increasing its length more than that of the fibula; (2) miniaturizations of the limb reduce both tibiotarsus and fibula length, but are reglarly associated with structural reductions of the distal parts of the fibula. True structrual reductions are distinguished from relative size reductions. The specific features of fibula reduction are analyzed through experimental mesenchyme excisions in chick limb buds. The methodical variation of experimental parameters resolves a long‐standing controversy about the effects of mesenchyme reductions on the patterns of skeletal formation. Mesenchyme excisions are shown to have unequal effects on the two zeugopod bones, affecting the fibula to a greater degree than the tibiotarsus. Several of the features seen in birds with advanced fibula reductions are paralleled by the effects of mesenchyme reductions. The consequences of this differential susceptibility of the skeletal blastemata are discussed both in terms of pattern formation in limb development and in terms of its bearing on the patterns of evolutionary limb reduction. It is concluded that thresholds of cell number and blastema size in development constrain the patterns of phenotypic variation in avian limbs.

Evidence that the extraocular motor nuclei innervate monkey palisade endings

Research highlights ▶ Neuronal tracer was injected into the abducens nucleus of monkey. ▶ Tracer positive axons were observed in the lateral rectus muscles. ▶ Tracer positive axons were cholinergic and established motor terminals. ▶ Palisade endings in the lateral rectus contained tracer as well. ▶ This study shows that palisade endings originate from motor nuclei.

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.

Multiscale fractal characterization of three-dimensional gene expression data

This article reports on the application of the recently introduced concept of multiscale fractal dimension (MFD) as a resource for quantifying three-dimensional gene expression patterns in embryonic development. While traditional fractal dimensions provide interesting possibilities for quantifying pattern complexity, as defined by the intensity in which the pattern interacts with its surrounding space, those approaches fail to take into account the important fact that natural objects are not perfectly self-similar. By expressing the fractal behavior explicitly in terms of the spatial scale, the MFD provides a more comprehensive and objective characterization of the complexity of natural data such as gene expression profiles. After presenting the MFD concept as well as a technique for its numerical estimation, the potential of this measure for objectively quantifying gene expression is discussed, and a complete example is provided regarding the three-dimensional expression of the myogenic marker gene Myf5 along successive somites in a mouse embryo. In this specific case, the adopted technique proved itself a useful means for identifying spatial variations of gene expression intensity.