ABSTRACT
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
The complex shape of embryonic cartilage represents a true challenge for phenotyping and basic understanding of skeletal development. X-ray computed microtomography (μCT) enables inspecting relevant ...tissues in all three dimensions; however, most 3D models are still created by manual segmentation, which is a time-consuming and tedious task. In this work, we utilised a convolutional neural network (CNN) to automatically segment the most complex cartilaginous system represented by the developing nasal capsule. The main challenges of this task stem from the large size of the image data (over a thousand pixels in each dimension) and a relatively small training database, including genetically modified mouse embryos, where the phenotype of the analysed structures differs from the norm. We propose a CNN-based segmentation model optimised for the large image size that we trained using a unique manually annotated database. The segmentation model was able to segment the cartilaginous nasal capsule with a median accuracy of 84.44% (Dice coefficient). The time necessary for segmentation of new samples shortened from approximately 8 h needed for manual segmentation to mere 130 s per sample. This will greatly accelerate the throughput of μCT analysis of cartilaginous skeletal elements in animal models of developmental diseases.
Amyloid plaques are small (~ 50 μm), highly-dense aggregates of amyloid beta (Aβ) protein in brain tissue, supposed to play a key role in pathogenesis of Alzheimer's disease (AD). Plaques´ in vivo ...detection, spatial distribution and quantitative characterization could be an essential marker in diagnostics and evaluation of AD progress. However, current imaging methods in clinics possess substantial limits in sensitivity towards Aβ plaques to play a considerable role in AD screening. Contrast enhanced X-ray micro computed tomography (micro CT) is an emerging highly sensitive imaging technique capable of high resolution visualization of rodent brain. In this study we show the absorption based contrast enhanced X-ray micro CT imaging is viable method for detection and 3D analysis of Aβ plaques in transgenic rodent models of Alzheimer's disease. Using iodine contrasted brain tissue isolated from the Tg-F344-AD rat model we show the micro CT imaging is capable of precise imaging of Aβ plaques, making possible to further analyze various aspects of their 3D spatial distribution and other properties.
In vertebrates, head and trunk muscles develop from different mesodermal populations and are regulated by distinct genetic networks. Neck muscles at the head-trunk interface remain poorly defined due ...to their complex morphogenesis and dual mesodermal origins. Here, we use genetically modified mice to establish a 3D model that integrates regulatory genes, cell populations and morphogenetic events that define this transition zone. We show that the evolutionary conserved cucullaris-derived muscles originate from posterior cardiopharyngeal mesoderm, not lateral plate mesoderm, and we define new boundaries for neural crest and mesodermal contributions to neck connective tissue. Furthermore, lineage studies and functional analysis of
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null mice reveal a unique developmental program for somitic neck muscles that is distinct from that of somitic trunk muscles. Our findings unveil the embryological and developmental requirements underlying tetrapod neck myogenesis and provide a blueprint to investigate how muscle subsets are selectively affected in some human myopathies.
Phase contrast imaging (PCI) is used to extend X-ray computed nanotomography (nCT) technique for analyzing samples with a low X-ray contrast, such as polymeric structures or soft tissues. Although ...this technique is used in many variations at synchrotrons, along with the development of X-ray tubes and X-ray detectors, a PCI becomes available also for laboratory systems. This paper is focused on determining the conditions for propagation-based PCI in laboratory nCT systems based on three criteria. It is mostly employed in near-field imaging regime, which is quantified via the Fresnel number. X-rays must reach a certain degree of coherence to form edge enhancement. Finally, the setup of every computed tomography (CT) measurement has to avoid geometrical unsharpness due to the finite focal spot size. These conditions are evaluated and discussed in terms of different properties and settings of the CT machine. Propagation-based PCI is tested on a sample of carbon fibers reinforced polyethylene and the implementation of the Paganin phase retrieval algorithm on the CT data is shown.
The imitation switch nuclear ATPase Smarca5 (Snf2h) is one of the most conserved chromatin remodeling factors. It exists in a variety of oligosubunit complexes that move DNA with respect to the ...histone octamer to generate regularly spaced nucleosomal arrays. Smarca5 interacts with different accessory proteins and represents a molecular motor for DNA replication, repair, and transcription. We deleted Smarca5 at the onset of definitive hematopoiesis (Vav1‐iCre) and observed that animals die during late fetal development due to anemia. Hematopoietic stem and progenitor cells accumulated but their maturation toward erythroid and myeloid lineages was inhibited. Proerythroblasts were dysplastic while basophilic erythroblasts were blocked in G2/M and depleted. Smarca5 deficiency led to increased p53 levels, its activation at two residues, one associated with DNA damage (S15Ph°s) second with CBP/p300 (K376Ac), and finally activation of the p53 targets. We also deleted Smarca5 in committed erythroid cells (Epor‐iCre) and observed that animals were anemic postnatally. Furthermore, 4‐hydroxytamoxifen‐mediated deletion of Smarca5 in the ex vivo cultures confirmed its requirement for erythroid cell proliferation. Thus, Smarca5 plays indispensable roles during early hematopoiesis and erythropoiesis. Stem Cells 2017;35:1614–1623
Smarca5 requirement during mouse hematopoietic development. Hematopoietic stem cells (HSCs) lacking Smarca5 temporarily activate proliferation followed by G2/M arrest during erythroid and myeloid differentiation leading to fatal anemia with tetraploid erythroblasts and p53 activation. Erythroid‐specific deletion of Smarca5 resulted in milder phenotypes: anemia and tetraploidy.
ISWI chromatin remodeling ATPase SMARCA5 (SNF2H) is a well-known factor for its role in regulation of DNA access via nucleosome sliding and assembly. SMARCA5 transcriptionally inhibits the myeloid ...master regulator PU.1. Upregulation of SMARCA5 was previously observed in CD34+ hematopoietic progenitors of acute myeloid leukemia (AML) patients. Since high levels of SMARCA5 are necessary for intensive cell proliferation and cell cycle progression of developing hematopoietic stem and progenitor cells in mice, we reasoned that removal of SMARCA5 enzymatic activity could affect the cycling or undifferentiated state of leukemic progenitor-like clones. Indeed, we observed that CRISPR/cas9-mediated
knockout in AML cell lines (S5KO) inhibited the cell cycle progression. We also observed that the
deletion induced karyorrhexis and nuclear budding as well as increased the ploidy, indicating its role in mitotic division of AML cells. The cytogenetic analysis of S5KO cells revealed the premature chromatid separation. We conclude that deleting SMARCA5 in AML blocks leukemic proliferation and chromatid cohesion.
Formation of oriented myofibrils is a key event in musculoskeletal development. However, the mechanisms that drive myocyte orientation and fusion to control muscle directionality in adults remain ...enigmatic. Here, we demonstrate that the developing skeleton instructs the directional outgrowth of skeletal muscle and other soft tissues during limb and facial morphogenesis in zebrafish and mouse. Time-lapse live imaging reveals that during early craniofacial development, myoblasts condense into round clusters corresponding to future muscle groups. These clusters undergo oriented stretch and alignment during embryonic growth. Genetic perturbation of cartilage patterning or size disrupts the directionality and number of myofibrils in vivo. Laser ablation of musculoskeletal attachment points reveals tension imposed by cartilage expansion on the forming myofibers. Application of continuous tension using artificial attachment points, or stretchable membrane substrates, is sufficient to drive polarization of myocyte populations in vitro. Overall, this work outlines a biomechanical guidance mechanism that is potentially useful for engineering functional skeletal muscle.
Organ function depends on tissues adopting the correct architecture. However, insights into organ architecture are currently hampered by an absence of standardized quantitative 3D analysis. We aimed ...to develop a robust technology to visualize, digitalize, and segment the architecture of two tubular systems in 3D:
o
ble resin
asting micro computed
omography (DUCT). As proof of principle, we applied DUCT to a mouse model for Alagille syndrome (
mice), characterized by intrahepatic bile duct paucity, that can spontaneously generate a biliary system in adulthood. DUCT identified increased central biliary branching and peripheral bile duct tortuosity as two compensatory processes occurring in distinct regions of
liver, leading to full reconstitution of wild-type biliary volume and phenotypic recovery. DUCT is thus a powerful new technology for 3D analysis, which can reveal novel phenotypes and provide a standardized method of defining liver architecture in mouse models.
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.
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