Tomáš Zikmund

Thyroid Hormone Receptor α Mutation Causes a Severe and Thyroxine-Resistant Skeletal Dysplasia in Female Mice

A new genetic disorder has been identified that results from mutation of THRA, encoding thyroid hormone receptor α1 (TRα1). Affected children have a high serum T3:T4 ratio and variable degrees of intellectual deficit and constipation but exhibit a consistently severe skeletal dysplasia. In an attempt to improve developmental delay and alleviate symptoms of hypothyroidism, patients are receiving varying doses and durations of T4 treatment, but responses have been inconsistent so far. Thra1PV/+ mice express a similar potent dominant-negative mutant TRα1 to affected individuals, and thus represent an excellent disease model. We hypothesized that Thra1PV/+ mice could be used to predict the skeletal outcome of human THRA mutations and determine whether prolonged treatment with a supraphysiological dose of T4 ameliorates the skeletal abnormalities. Adult female Thra1PV/+ mice had short stature, grossly abnormal bone morphology but normal bone strength despite high bone mass. Although T4 treatment suppressed TSH secretion, it had no effect on skeletal maturation, linear growth, or bone mineralization, thus demonstrating profound tissue resistance to thyroid hormone. Despite this, prolonged T4 treatment abnormally increased bone stiffness and strength, suggesting the potential for detrimental consequences in the long term. Our studies establish that TRα1 has an essential role in the developing and adult skeleton and predict that patients with different THRA mutations will display variable responses to T4 treatment, which depend on the severity of the causative mutation.

Superficial cells are self-renewing chondrocyte progenitors, which form the articular cartilage in juvenile mice.

Articular cartilage has little regenerative capacity. Recently, genetic lineage tracing experiments have revealed chondrocyte progenitors at the articular surface. We further characterized these progenitors by using in vivo genetic approaches. Histone H2B–green fluorescent protein retention revealed that superficial cells divide more slowly than underlying articular chondrocytes. Clonal genetic tracing combined with immunohistochemistry revealed that superficial cells renew their number by symmetric division, express mesenchymal stem cell markers, and generate chondrocytes via both asymmetric and symmetric differentiation. Quantitative analysis of cellular kinetics, in combination with phosphotungstic acid–enhanced micro–computed tomography, showed that superficial cells generate chondrocytes and contribute to the growth and reshaping of articular cartilage. Furthermore, we found that cartilage renewal occurs as the progeny of superficial cells fully replace fetal chondrocytes during early postnatal life. Thus, superficial cells are self‐renewing progenitors that are capable of maintaining their own population and fulfilling criteria of unipotent adult stem cells. Furthermore, the progeny of these cells reconstitute adult articular cartilage de novo, entirely substituting fetal chondrocytes.—Li, L., Newton, P. T., Bouderlique, T., Sejnohova, M., Zikmund, T., Kozhemyakina, E., Xie, M., Krivanek, J., Kaiser, J., Qian, H., Dyachuk, V., Lassar, A. B., Warman, M. L., Barenius, B., Adameyko, I., Chagin, A. S. Superficial cells are self‐renewing chondrocyte progenitors, which form the articular cartilage in juvenile mice. FASEB J. 31, 1067–1084 (2017). www.fasebj.org

Analysis of neural crest–derived clones reveals novel aspects of facial development

Facial shaping results from oriented divisions and crowd movements of ectomesenchymal cells during morphogenetic events. Cranial neural crest cells populate the future facial region and produce ectomesenchyme-derived tissues, such as cartilage, bone, dermis, smooth muscle, adipocytes, and many others. However, the contribution of individual neural crest cells to certain facial locations and the general spatial clonal organization of the ectomesenchyme have not been determined. We investigated how neural crest cells give rise to clonally organized ectomesenchyme and how this early ectomesenchyme behaves during the developmental processes that shape the face. Using a combination of mouse and zebrafish models, we analyzed individual migration, cell crowd movement, oriented cell division, clonal spatial overlapping, and multilineage differentiation. The early face appears to be built from multiple spatially defined overlapping ectomesenchymal clones. During early face development, these clones remain oligopotent and generate various tissues in a given location. By combining clonal analysis, computer simulations, mouse mutants, and live imaging, we show that facial shaping results from an array of local cellular activities in the ectomesenchyme. These activities mostly involve oriented divisions and crowd movements of cells during morphogenetic events. Cellular behavior that can be recognized as individual cell migration is very limited and short-ranged and likely results from cellular mixing due to the proliferation activity of the tissue. These cellular mechanisms resemble the strategy behind limb bud morphogenesis, suggesting the possibility of common principles and deep homology between facial and limb outgrowth.

On fragmenting, densely mineralised acellular protrusions into articular cartilage and their possible role in osteoarthritis.

High density mineralised protrusions (HDMP) from the tidemark mineralising front into hyaline articular cartilage (HAC) were first described in Thoroughbred racehorse fetlock joints and later in Icelandic horse hock joints. We now report them in human material. Whole femoral heads removed at operation for joint replacement or from dissection room cadavers were imaged using magnetic resonance imaging (MRI) dual echo steady state at 0.23 mm resolution, then 26‐μm resolution high contrast X‐ray microtomography, sectioned and embedded in polymethylmethacrylate, blocks cut and polished and re‐imaged with 6‐μm resolution X‐ray microtomography. Tissue mineralisation density was imaged using backscattered electron SEM (BSE SEM) at 20 kV with uncoated samples. HAC histology was studied by BSE SEM after staining block faces with ammonium triiodide solution. HDMP arise via the extrusion of an unknown mineralisable matrix into clefts in HAC, a process of acellular dystrophic calcification. Their formation may be an extension of a crack self‐healing mechanism found in bone and articular calcified cartilage. Mineral concentration exceeds that of articular calcified cartilage and is not uniform. It is probable that they have not been reported previously because they are removed by decalcification with standard protocols. Mineral phase morphology frequently shows the agglomeration of many fine particles into larger concretions. HDMP are surrounded by HAC, are brittle, and show fault lines within them. Dense fragments found within damaged HAC could make a significant contribution to joint destruction. At least larger HDMP can be detected with the best MRI imaging ex vivo.

Oriented clonal cell dynamics enables accurate growth and shaping of vertebrate cartilage

Cartilaginous structures are at the core of embryo growth and shaping before the bone forms. Here we report a novel principle of vertebrate cartilage growth that is based on introducing transversally-oriented clones into pre-existing cartilage. This mechanism of growth uncouples the lateral expansion of curved cartilaginous sheets from the control of cartilage thickness, a process which might be the evolutionary mechanism underlying adaptations of facial shape. In rod-shaped cartilage structures (Meckel, ribs and skeletal elements in developing limbs), the transverse integration of clonal columns determines the well-defined diameter and resulting rod-like morphology. We were able to alter cartilage shape by experimentally manipulating clonal geometries. Using in silico modeling, we discovered that anisotropic proliferation might explain cartilage bending and groove formation at the macro-scale. DOI: http://dx.doi.org/10.7554/eLife.25902.001

Use of micro computed-tomography and 3D printing for reverse engineering of mouse embryo nasal capsule

Imaging of increasingly complex cartilage in vertebrate embryos is one of the key tasks of developmental biology. This is especially important to study shape-organizing processes during initial skeletal formation and growth. Advanced imaging techniques that are reflecting biological needs give a powerful impulse to push the boundaries of biological visualization. Recently, techniques for contrasting tissues and organs have improved considerably, extending traditional 2D imaging approaches to 3D . X-ray micro computed tomography (μCT), which allows 3D imaging of biological objects including their internal structures with a resolution in the micrometer range, in combination with contrasting techniques seems to be the most suitable approach for non-destructive imaging of embryonic developing cartilage. Despite there are many software-based ways for visualization of 3D data sets, having a real solid model of the studied object might give novel opportunities to fully understand the shape-organizing processes in the developing body. In this feasibility study we demonstrated the full procedure of creating a real 3D object of mouse embryo nasal capsule, i.e. the staining, the μCT scanning combined by the advanced data processing and the 3D printing.

Sequential processing of quantitative phase images for the study of cell behaviour in real‐time digital holographic microscopy

Transmitted light holographic microscopy is particularly used for quantitative phase imaging of transparent microscopic objects such as living cells. The study of the cell is based on extraction of the dynamic data on cell behaviour from the time‐lapse sequence of the phase images. However, the phase images are affected by the phase aberrations that make the analysis particularly difficult. This is because the phase deformation is prone to change during long‐term experiments. Here, we present a novel algorithm for sequential processing of living cells phase images in a time‐lapse sequence. The algorithm compensates for the deformation of a phase image using weighted least‐squares surface fitting. Moreover, it identifies and segments the individual cells in the phase image. All these procedures are performed automatically and applied immediately after obtaining every single phase image. This property of the algorithm is important for real‐time cell quantitative phase imaging and instantaneous control of the course of the experiment by playback of the recorded sequence up to actual time. Such operators intervention is a forerunner of process automation derived from image analysis. The efficiency of the propounded algorithm is demonstrated on images of rat fibrosarcoma cells using an off‐axis holographic microscope.

Accurate micro-computed tomography imaging of pore spaces in collagen-based scaffold

In this work we have used X-ray micro-computed tomography (μCT) as a method to observe the morphology of 3D porous pure collagen and collagen-composite scaffolds useful in tissue engineering. Two aspects of visualizations were taken into consideration: improvement of the scan and investigation of its sensitivity to the scan parameters. Due to the low material density some parts of collagen scaffolds are invisible in a μCT scan. Therefore, here we present different contrast agents, which increase the contrast of the scanned biopolymeric sample for μCT visualization. The increase of contrast of collagenous scaffolds was performed with ceramic hydroxyapatite microparticles (HAp), silver ions (Ag+) and silver nanoparticles (Ag-NPs). Since a relatively small change in imaging parameters (e.g. in 3D volume rendering, threshold value and μCT acquisition conditions) leads to a completely different visualized pattern, we have optimized these parameters to obtain the most realistic picture for visual and qualitative evaluation of the biopolymeric scaffold. Moreover, scaffold images were stereoscopically visualized in order to better see the 3D biopolymer composite scaffold morphology. However, the optimized visualization has some discontinuities in zoomed view, which can be problematic for further analysis of interconnected pores by commonly used numerical methods. Therefore, we applied the locally adaptive method to solve discontinuities issue. The combination of contrast agent and imaging techniques presented in this paper help us to better understand the structure and morphology of the biopolymeric scaffold that is crucial in the design of new biomaterials useful in tissue engineering.

Dynamic phase differences based on quantitative phase imaging for the objective evaluation of cell behavior.

Abstract. Quantitative phase imaging (QPI) brought innovation to noninvasive observation of live cell dynamics seen as cell behavior. Unlike the Zernike phase contrast or differential interference contrast, QPI provides quantitative information about cell dry mass distribution. We used such data for objective evaluation of live cell behavioral dynamics by the advanced method of dynamic phase differences (DPDs). The DPDs method is considered a rational instrument offered by QPI. By subtracting the antecedent from the subsequent image in a time-lapse series, only the changes in mass distribution in the cell are detected. The result is either visualized as a two-dimensional color-coded projection of these two states of the cell or as a time dependence of changes quantified in picograms. Then in a series of time-lapse recordings, the chain of cell mass distribution changes that would otherwise escape attention is revealed. Consequently, new salient features of live cell behavior should emerge. Construction of the DPDs method and results exhibiting the approach are presented. Advantage of the DPDs application is demonstrated on cells exposed to an osmotic challenge. For time-lapse acquisition of quantitative phase images, the recently developed coherence-controlled holographic microscope was employed.

Frontoparietal Bone in Extinct Palaeobatrachidae (Anura): Its Variation and Taxonomic Value.

Palaeobatrachidae are extinct frogs from Europe closely related to the Gondwanan Pipidae, which includes Xenopus. Their frontoparietal is a distinctive skeletal element which has served as a basis for establishing the genus Albionbatrachus. Because little was known about developmental and individual variation of the frontoparietal, and its usefulness in delimiting genera and species has sometimes been doubted, we investigate its structure in Palaeobatrachus and Albionbatrachus by means of X‐ray high resolution computer tomography (micro‐CT). To infer the scope of variation present in the fossil specimens, we also examined developmental and interspecific variation in extant Xenopus. In adults of extinct taxa, the internal structure of the frontoparietal bone consists of a superficial and a basal layer of compact bone, with a middle layer of cancellous bone between them, much as in early amphibians. In Albionbatrachus, the layer of cancellous bone, consisting of small and large cavities, was connected with the dorsal, sculptured surface of the bone by a system of narrow canals; in Palaeobatrachus, the layer of cancellous bone and the canals connecting this layer with the dorsal surface of the frontoparietal were reduced. The situation in Palaeobatrachus robustus from the lower Miocene of France is intermediate—while external features support assignment to Palaeobatrachus, the inner structure is similar to that in Albionbatrachus. It may be hypothesized that sculptured frontoparietals with a well‐developed layer of cancellous (i.e., vascularized) bone may indicate adaptation to a more terrestrial way of life, whereas a reduced cancellous layer might indicate a permanent water dweller. Anat Rec, 298:1848–1863, 2015.