Ronald Seidel

Ultrastructural and developmental features of the tessellated endoskeleton of elasmobranchs (sharks and rays)

The endoskeleton of elasmobranchs (sharks and rays) is comprised largely of unmineralized cartilage, differing fundamentally from the bony skeletons of other vertebrates. Elasmobranch skeletons are further distinguished by a tessellated surface mineralization, a layer of minute, polygonal, mineralized tiles called tesserae. This ‘tessellation’ has defined the elasmobranch group for more than 400 million years, yet the limited data on development and ultrastructure of elasmobranch skeletons (e.g. how tesserae change in shape and mineral density with age) have restricted our abilities to develop hypotheses for tessellated cartilage growth. Using high‐resolution, two‐dimensional and three‐dimensional materials and structural characterization techniques, we investigate an ontogenetic series of tessellated cartilage from round stingray Urobatis halleri, allowing us to define a series of distinct phases for skeletal mineralization and previously unrecognized features of tesseral anatomy. We show that the distinct tiled morphology of elasmobranch calcified cartilage is established early in U. halleri development, with tesserae forming first in histotroph embryos as isolated, globular islets of mineralized tissue. By the sub‐adult stage, tesserae have increased in size and grown into contact with one another. The intertesseral contact results in the formation of more geometric (straight‐edged) tesseral shapes and the development of two important features of tesseral anatomy, which we describe here for the first time. The first, the intertesseral joint, where neighboring tesserae abut without appreciable overlapping or interlocking, is far more complex than previously realized, comprised of a convoluted bearing surface surrounded by areas of fibrous attachment. The second, tesseral spokes, are lamellated, high‐mineral density features radiating outward, like spokes on a wheel, from the center of each tessera to its joints with its neighbors, likely acting as structural reinforcements of the articulations between tesserae. As tesserae increase in size during ontogeny, spokes are lengthened via the addition of new lamellae, resulting in a visually striking mineralization pattern in the larger tesserae of older adult skeletons when viewed with scanning electron microscopy (SEM) in backscatter mode. Backscatter SEM also revealed that the cell lacunae in the center of larger tesserae are often filled with high mineral density material, suggesting that when intratesseral cells die, cell‐regulated inhibition of mineralization is interrupted. Many of the defining ultrastructural details we describe relate to local variation in tissue mineral density and support previously proposed accretive growth mechanisms for tesserae. High‐resolution micro‐computed tomography data indicate that some tesseral anatomical features we describe for U. halleri are common among species of all major elasmobranch groups despite large variation in tesseral shape and size. We discuss hypotheses about how these features develop, and compare them with other vertebrate skeletal tissue types and their growth mechanisms.

Overcoming the fragility – X-ray computed micro-tomography elucidates brachiopod endoskeletons

IntroductionThe calcareous shells of brachiopods offer a wealth of informative characters for taxonomic and phylogenetic investigations. In particular scanning electron microscopy (SEM) has been used for decades to visualise internal structures of the shell. However, to produce informative SEM data, brachiopod shells need to be opened after chemical removal of the soft tissue. This preparation occasionally damages the shell. Additionally, skeletal elements of taxonomic/systematic interest such as calcareous spicules which are loosely embedded in the lophophore and mantle connective tissue become disintegrated during the preparation process.ResultsUsing a nondestructive micro-computed tomography (μCT) approach, the entire fragile endoskeleton of brachiopods is documented for the first time. New insights on the structure and position of tissue-bound skeletal elements (spicules) are given as add ons to existing descriptions of brachiopod shell anatomy, thereby enhancing the quality and quantity of informative characters needed for both taxonomic and phylogenetic studies. Here, we present five modern, articulated brachiopods (Rectocalathis schemmgregoryi n. gen., n. sp., Eucalathis sp., Gryphus vitreus, Liothyrella neozelanica and Terebratulina retusa) that were X-rayed using a Phoenix Nanotom XS 180 NF. We provide links to download 3D models of these species, and additional five species with spicules can be accessed in the Supplemental Material. In total, 17 brachiopod genera covering all modern articulated subgroups and 2 inarticulated genera were X-rayed for morphological analysis. Rectocalathis schemmgregoryi n. gen., n. sp. is fully described.ConclusionMicro-CT is an excellent non-destructive tool for investigating calcified structures in the exo- and endoskeletons of brachiopods. With high quality images and interactive 3D models, this study provides a comprehensive description of the profound differences in shell anatomy, facilitates the detection of new delicate morphological characters of the endoskeleton and gives new insights into the body plan of modern brachiopods.

Automated segmentation of complex patterns in biological tissues: Lessons from stingray tessellated cartilage

Introduction Many biological structures show recurring tiling patterns on one structural level or the other. Current image acquisition techniques are able to resolve those tiling patterns to allow quantitative analyses. The resulting image data, however, may contain an enormous number of elements. This renders manual image analysis infeasible, in particular when statistical analysis is to be conducted, requiring a larger number of image data to be analyzed. As a consequence, the analysis process needs to be automated to a large degree. In this paper, we describe a multi-step image segmentation pipeline for the automated segmentation of the calcified cartilage into individual tesserae from computed tomography images of skeletal elements of stingrays. Methods Besides applying state-of-the-art algorithms like anisotropic diffusion smoothing, local thresholding for foreground segmentation, distance map calculation, and hierarchical watershed, we exploit a graph-based representation for fast correction of the segmentation. In addition, we propose a new distance map that is computed only in the plane that locally best approximates the calcified cartilage. This distance map drastically improves the separation of individual tesserae. We apply our segmentation pipeline to hyomandibulae from three individuals of the round stingray (Urobatis halleri), varying both in age and size. Results Each of the hyomandibula datasets contains approximately 3000 tesserae. To evaluate the quality of the automated segmentation, four expert users manually generated ground truth segmentations of small parts of one hyomandibula. These ground truth segmentations allowed us to compare the segmentation quality w.r.t. individual tesserae. Additionally, to investigate the segmentation quality of whole skeletal elements, landmarks were manually placed on all tesserae and their positions were then compared to the segmented tesserae. With the proposed segmentation pipeline, we sped up the processing of a single skeletal element from days or weeks to a few hours.