Caroline Morgenthaler

Comparison of five different RNA sources to examine the lactating bovine mammary gland transcriptome using RNA-Sequencing.

The objective of this study was to examine five different sources of RNA, namely mammary gland tissue (MGT), milk somatic cells (SC), laser microdissected mammary epithelial cells (LCMEC), milk fat globules (MFG) and antibody-captured milk mammary epithelial cells (mMEC) to analyze the bovine mammary gland transcriptome using RNA-Sequencing. Our results provide a comparison between different sampling methods (invasive and non-invasive) to define the transcriptome of mammary gland tissue and milk cells. This information will be of value to investigators in choosing the most appropriate sampling method for different research applications to study specific physiological states during lactation. One of the simplest procedures to study the transcriptome associated with milk appears to be the isolation of total RNA directly from SC or MFG released into milk during lactation. Our results indicate that the SC and MFG transcriptome are representative of MGT and LCMEC and can be used as effective and alternative samples to study mammary gland expression without the need to perform a tissue biopsy.

Chondrocytes Play a Major Role in the Stimulation of Bone Growth by Thyroid Hormone

Thyroid hormone (T3) is required for postnatal skeletal growth. It exerts its effect by binding to nuclear receptors, TRs including TRα1 and TRβ1, which are present in most cell types. These cell types include chondrocytes and osteoblasts, the interactions of which are known to regulate endochondral bone formation. In order to analyze the respective functions of T3 stimulation in chondrocytes and osteoblasts during postnatal growth, we use Cre/loxP recombination to express a dominant-negative TRα1(L400R) mutant receptor in a cell-specific manner. Phenotype analysis revealed that inhibiting T3 response in chondrocytes is sufficient to reproduce the defects observed in hypothyroid mice, not only for cartilage maturation, but also for ossification and mineralization. TRα1(L400R) in chondrocytes also results in skull deformation. In the meantime, TRα1(L400R) expression in mature osteoblasts has no visible effect. Transcriptome analysis identifies a number of changes in gene expression induced by TRα1(L400R) in cartilage. These changes suggest that T3 normally cross talks with several other signaling pathways to promote chondrocytes proliferation, differentiation, and skeletal growth.

Integrated mRNA and miRNA expression profiling in blood reveals candidate biomarkers associated with endurance exercise in the horse

The adaptive response to extreme endurance exercise might involve transcriptional and translational regulation by microRNAs (miRNAs). Therefore, the objective of the present study was to perform an integrated analysis of the blood transcriptome and miRNome (using microarrays) in the horse before and after a 160 km endurance competition. A total of 2,453 differentially expressed genes and 167 differentially expressed microRNAs were identified when comparing pre- and post-ride samples. We used a hypergeometric test and its generalization to gain a better understanding of the biological functions regulated by the differentially expressed microRNA. In particular, 44 differentially expressed microRNAs putatively regulated a total of 351 depleted differentially expressed genes involved variously in glucose metabolism, fatty acid oxidation, mitochondrion biogenesis, and immune response pathways. In an independent validation set of animals, graphical Gaussian models confirmed that miR-21-5p, miR-181b-5p and miR-505-5p are candidate regulatory molecules for the adaptation to endurance exercise in the horse. To the best of our knowledge, the present study is the first to provide a comprehensive, integrated overview of the microRNA-mRNA co-regulation networks that may have a key role in controlling post-transcriptomic regulation during endurance exercise.

Prnp knockdown in transgenic mice using RNA interference

RNA interference has become a widely used approach to perform gene knockdown experiments in cell cultures and more recently transgenic animals. A designed miRNA targeting the prion protein mRNA was built and expressed using the human PRNP promoter. Its efficiency was confirmed in transfected cells and it was used to generate several transgenic mouse lines. Although expressed at low levels, it was found to downregulate the endogenous mouse Prnp gene expression to an extent that appears to be directly related with the transgene expression level and that could reach up to 80% inhibition. This result highlights the potential and limitations of the RNA interference approach when applied to disease resistance.

Omics technologies provide new insights into the molecular physiopathology of equine osteochondrosis

BackgroundOsteochondrosis (OC(D)) is a juvenile osteo-articular disorder affecting several mammalian species. In horses, OC(D) is considered as a multifactorial disease and has been described as a focal disruption of endochondral ossification leading to the development of osteoarticular lesions. Nevertheless, OC(D) physiopathology is poorly understood. Affected horses may present joint swelling, stiffness and lameness. Thus, OC(D) is a major concern for the equine industry. Our study was designed as an integrative approach using omics technologies for the identification of constitutive defects in epiphyseal cartilage and/or subchondral bone associated with the development of primary lesions to further understand OC(D) pathology. This study compared samples from non-affected joints (hence lesion-free) from OC(D)-affected foals (n = 5, considered predisposed samples) with samples from OC-free foals (n = 5) considered as control samples. Consequently, results are not confounded by changes associated with the evolution of the lesion, but focus on altered constitutive molecular mechanisms. Comparative proteomics and micro computed tomography analyses were performed on predisposed and OC-free bone and cartilage samples. Metabolomics was also performed on synovial fluid from OC-free, OC(D)-affected and predisposed joints.ResultsTwo lesion subtypes were identified: OCD (lesion with fragment) and OC (osteochondral defects). Modulated proteins were identified using omics technologies (2-DE proteomics) in cartilage and bone from affected foals compare to OC-free foals. These were associated with cellular processes including cell cycle, energy production, cell signaling and adhesion as well as tissue-specific processes such as chondrocyte maturation, extracellular matrix and mineral metabolism. Of these, five had already been identified in synovial fluid of OC-affected foals: ACTG1 (actin, gamma 1), albumin, haptoglobin, FBG (fibrinogen beta chain) and C4BPA (complement component 4 binding protein, alpha).ConclusionThis study suggests that OCD lesions may result from a cartilage defect whereas OC lesions may be triggered by both bone and cartilage defects, suggesting that different molecular mechanisms responsible for the equine osteochondrosis lesion subtypes and predisposition could be due to a defect in both bone and cartilage. This study will contribute to refining the definition of OC(D) lesions and may improve diagnosis and development of therapies for horses and other species, including humans.

Endurance Exercise Ability in the Horse: A Trait with Complex Polygenic Determinism

Endurance horses are able to run at more than 20 km/h for 160 km (in bouts of 30–40 km). This level of performance is based on intense aerobic metabolism, effective body heat dissipation and the ability to endure painful exercise. The known heritabilities of endurance performance and exercise-related physiological traits in Arabian horses suggest that adaptation to extreme endurance exercise is influenced by genetic factors. The objective of the present genome-wide association study (GWAS) was to identify single nucleotide polymorphisms (SNPs) related to endurance racing performance in 597 Arabian horses. The performance traits studied were the total race distance, average race speed and finishing status (qualified, eliminated or retired). We used three mixed models that included a fixed allele or genotype effect and a random, polygenic effect. Quantile-quantile plots were acceptable, and the regression coefficients for actual vs. expected log10 p-values ranged from 0.865 to 1.055. The GWAS revealed five significant quantitative trait loci (QTL) corresponding to 6 SNPs on chromosomes 6, 1, 7, 16, and 29 (two SNPs) with corrected p-values from 1.7 × 10−6 to 1.8 × 10−5. Annotation of these 5 QTL revealed two genes: sortilin-related VPS10-domain-containing receptor 3 (SORCS3) on chromosome 1 is involved in protein trafficking, and solute carrier family 39 member 12 (SLC39A12) on chromosome 29 is active in zinc transport and cell homeostasis. These two coding genes could be involved in neuronal tissues (CNS). The other QTL on chromosomes 6, 7, and 16 may be involved in the regulation of the gene expression through non-coding RNAs, CpG islands and transcription factor binding sites. On chromosome 6, a new candidate equine long non-coding RNA (KCNQ1OT1 ortholog: opposite antisense transcript 1 of potassium voltage-gated channel subfamily Q member 1 gene) was predicted in silico and validated by RT-qPCR in primary cultures of equine myoblasts and fibroblasts. This lncRNA could be one element of the cardiac rhythm regulation. Our GWAS revealed that equine performance during endurance races is a complex polygenic trait, and is partially governed by at least 5 QTL: two coding genes involved in neuronal tissues and three other loci with many regulatory functions such as slowing down heart rate.