Teleost fish such as
Danio rerio
(zebrafish) have been successfully used in biomedical research since decades. Genetically altered fish lines obtained by state-of-the-art genetic technologies are ...serving as well-known model organisms. In Europe, following Directive 2010/63/EU, generation, breeding, and husbandry of new genetically altered lines of laboratory animals require governmental state approval in case pain, suffering, distress, or long-lasting harm to the offspring derived by breeding of these lines cannot be excluded. The identification and assessment of pain, distress, or harm, according to a severity classification of mild, moderate, severe, or humane endpoint, became a new challenging task for all scientists, animal technicians, and veterinarians for daily work with laboratory zebrafish. In this study, we describe the performance of the assessment of welfare parameters of selected pathologic phenotypes and abnormalities frequently found in laboratory fish facilities based on veterinary, biological, and physiological aspects by using a dedicated score sheet. In a colony of zebrafish, we evaluated the frequency of genotype-independent abnormalities observed within 3 years. We give examples for severity classification and measures once an abnormality has been identified according to the 3Rs (Replacement, Reduction and Refinement).
ABSTRACT
Tendons are an essential part of the musculoskeletal system, connecting muscle and skeletal elements to enable force generation. The transcription factor scleraxis marks vertebrate tendons ...from early specification. Scleraxis‐null mice are viable and have a range of tendon and bone defects in the trunk and limbs but no described cranial phenotype. We report the expression of zebrafish scleraxis orthologs: scleraxis homolog (scx)‐a and scxb in cranial and intramuscular tendons and in other skeletal elements. Single mutants for either scxa or scxb, generated by clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR‐associated protein 9 (Cas9), are viable and fertile as adult fish. Although scxb mutants show no obvious phenotype, scxa mutant embryos have defects in cranial tendon maturation and muscle misalignment. Mutation of both scleraxis genes results in more severe defects in cranial tendon differentiation, muscle and cartilage dysmorphogenesis and paralysis, and lethality by 2–5 wk, which indicates an essential function of scleraxis for craniofacial development. At juvenile and adult stages, ribs in scxa mutants fail to mineralize and/or are small and heavily fractured. Scxa mutants also have smaller muscle volume, abnormal swim movement, and defects in bone growth and composition. Scleraxis function is therefore essential for normal craniofacial form and function and vital for fish development.—Kague, E., Hughes, S. M., Lawrence, E. A., Cross, S., Martin‐Silverstone, E., Hammond, C. L., Hinits, Y. Scleraxis genes are required for normal musculoskeletal development and for rib growth and mineralization in zebrafish. FASEB J. 33, 9116–9130 (2019). www.fasebj.org
Collagen is the major structural component of cartilage, and mutations in the genes encoding type XI collagen are associated with severe skeletal dysplasias (fibrochondrogenesis and Stickler ...syndrome) and early-onset osteoarthritis (OA). The impact of the lack of type XI collagen on cell behaviour and mechanical performance during skeleton development is unknown. We studied a zebrafish mutant for col11a2 and evaluated cartilage, bone development and mechanical properties to address this. We show that in col11a2 mutants, type II collagen is made but is prematurely degraded in maturing cartilage and ectopically expressed in the joint. These changes are correlated with increased stiffness of both bone and cartilage; quantified using atomic force microscopy. In the mutants, the skeletal rudiment terminal region in the jaw joint is broader and the interzone smaller. These differences in shape and material properties impact on joint function and mechanical performance, which we modelled using finite element analyses. Finally, we show that col11a2 heterozygous carriers reach adulthood but show signs of severe early-onset OA. Taken together, our data demonstrate a key role for type XI collagen in maintaining the properties of cartilage matrix; which when lost leads to alterations to cell behaviour that give rise to joint pathologies.
This article is part of the Theo Murphy meeting issue ‘Mechanics of development’.
The spine is the central skeletal support structure in vertebrates consisting of repeated units of bone, the vertebrae, separated by intervertebral discs (IVDs) that enable the movement of the spine. ...Spinal pathologies such as idiopathic back pain, vertebral compression fractures and IVD failure affect millions of people worldwide. Animal models can help us to understand the disease process, and zebrafish are increasingly used as they are highly genetically tractable, their spines are axially loaded like humans, and they show similar pathologies to humans during ageing. However, biomechanical models for the zebrafish are largely lacking. Here, we describe the results of loading intact zebrafish spinal motion segments on a material testing stage within a micro-computed tomography machine. We show that vertebrae and their arches show predictable patterns of deformation prior to their ultimate failure, in a pattern dependent on their position within the segment. We further show using geometric morphometrics which regions of the vertebra deform the most during loading, and that finite-element models of the trunk subjected reflect the real patterns of deformation and strain seen during loading and can therefore be used as a predictive model for biomechanical performance.
A more accurate understanding of the molecular mechanisms and signaling pathways underpinning human mesenchymal stem cell (MSC) plasticity and differentiation properties is pivotal for accomplishing ...solid and diligent translation of MSC-based experimental therapeutics and clinical trials to broad clinical practice. In addition, this knowledge enables selection of MSC subpopulations with increased differentiation potential and/or use of exogenous factors to boost this potential. Here, we report that CD105 (ENG) is a predictive biomarker of osteogenic potential in two types of MSCs: stem cells from human exfoliated deciduous teeth (SHED) and human adipose-derived stem cells (hASC). We also validate that CD105 can be used to select and enrich for subpopulations of SHED and hASC with higher in vitro osteogenic potential. In addition, we show that hsa-mir-1287 regulates CD105 expression, and propose that fine-tuning hsa-mir-1287 levels could be used to control osteopotential in SHED. These findings provide better discernment of the molecular bases behind MSC osteogenic plasticity and open up new perspectives to leverage osteogenic potential in MSCs by modulation of a specific miRNA.
Display omitted
Collagen is the major structural component of cartilage, and mutations in the genes encoding type XI collagen are associated with severe skeletal dysplasias (fibrochondrogenesis and Stickler ...syndrome) and early-onset osteoarthritis (OA). The impact of the lack of type XI collagen on cell behaviour and mechanical performance during skeleton development is unknown. We studied a zebrafish mutant for col11a2 and evaluated cartilage, bone development and mechanical properties to address this. We show that in col11a2 mutants, type II collagen is made but is prematurely degraded in maturing cartilage and ectopically expressed in the joint. These changes are correlated with increased stiffness of both bone and cartilage; quantified using atomic force microscopy. In the mutants, the skeletal rudiment terminal region in the jaw joint is broader and the interzone smaller. These differences in shape and material properties impact on joint function and mechanical performance, which we modelled using finite element analyses. Finally, we show that col11a2 heterozygous carriers reach adulthood but show signs of severe early-onset OA. Taken together, our data demonstrate a key role for type XI collagen in maintaining the properties of cartilage matrix; which when lost leads to alterations to cell behaviour that give rise to joint pathologies.
This article is part of the Theo Murphy meeting issue 'Mechanics of development'.
Gastric cancer is one of the top list of cancer types that most leads to death in Brazil and worldwide. Helicobacter pylori(H. pylori) is a class I carcinogen and infect almost 90% of chronic ...gastritis patients. Some genotypes confer different virulent potential to H. pylori and can increase the risk of gastritis development. Methylation of CpG islands can inactivate tumor suppressor genes and therefore, it can be involved in the tumorigenic process. CDH1 is a tumor suppressor gene that encodes the E-cadherin protein, which is important in maintaining cell-cell contacts. The inactivation of this gene can increase the chance of metastasis. Promoter methylation of CDH1 at early steps of gastric carcinogenesis is not yet completely understood.
In this study, we investigated the methylation status of CDH1 in chronic gastritis samples and correlated it with the presence of H. pylori.
Sixty gastric mucosal biopsies were used in this study. The detection of H. pylori was performed with the PCR primers specific to urease C gene. H. pylori genotyping was performed by PCR to cagA and vacA (s and m region). The methylation status of these gene CDH1 was analyzed using methylation-specific polymerase chain reaction and direct sequencing of the PCR products was performed using primers methylated and unmethylated in both forward and reverse directions.
H. pylori was detected in 90% of chronic gastritis samples; among these 33% were cagA positive and 100% vacA s1. The genotype vacA s2/m1 was not detected in any sample analyzed. Methylation of CDH1 was detected in 63.3% of chronic gastritis samples and 95% of them were also H. pylori-positive.
This work suggests that CDH1 gene methylation and H. pylori infection are frequent events in samples from Brazilian patients with chronic gastritis and reinforces the correlation between H. pylori infection and CDH1 inactivation in early steps of gastric tumorigenesis.
Abstract
Hearing loss is a frequent sensory impairment in humans and genetic factors account for an elevated fraction of the cases. We have investigated a large family of five generations, with 15 ...reported individuals presenting non-syndromic, sensorineural, bilateral and progressive hearing loss, segregating as an autosomal dominant condition. Linkage analysis, using SNP-array and selected microsatellites, identified a region of near 13 cM in chromosome 20 as the best candidate to harbour the causative mutation. After exome sequencing and filtering of variants, only one predicted deleterious variant in the NCOA3 gene (NM_181659, c.2810C > G; p.Ser937Cys) fit in with our linkage data. RT-PCR, immunostaining and in situ hybridization showed expression of ncoa3 in the inner ear of mice and zebrafish. We generated a stable homozygous zebrafish mutant line using the CRISPR/Cas9 system. ncoa3−/− did not display any major morphological abnormalities in the ear, however, anterior macular hair cells showed altered orientation. Surprisingly, chondrocytes forming the ear cartilage showed abnormal behaviour in ncoa3−/−, detaching from their location, invading the ear canal and blocking the cristae. Adult mutants displayed accumulation of denser material wrapping the otoliths of ncoa3−/− and increased bone mineral density. Altered zebrafish swimming behaviour corroborates a potential role of ncoa3 in hearing loss. In conclusion, we identified a potential candidate gene to explain hereditary hearing loss, and our functional analyses suggest subtle and abnormal skeletal behaviour as mechanisms involved in the pathogenesis of progressive sensory function impairment.
PDF
