Véronique Blanquet

Ubiquitous Gasp1 overexpression in mice leads mainly to a hypermuscular phenotype

BackgroundMyostatin, a member of the TGFβ superfamily, is well known as a potent and specific negative regulator of muscle growth. Targeting the myostatin signalling pathway may offer promising therapeutic strategies for the treatment of muscle-wasting disorders. In the last decade, various myostatin-binding proteins have been identified to be able to inhibit myostatin activity. One of these is GASP1 (Growth and Differentiation Factor-Associated Serum Protein-1), a protein containing a follistatin domain as well as multiple domains associated with protease inhibitors. Despite in vitro data, remarkably little is known about in vivo functions of Gasp1. To further address the role of GASP1 during mouse development and in adulthood, we generated a gain-of-function transgenic mouse model that overexpresses Gasp1 under transcriptional control of the human cytomegalovirus immediate-early promoter/enhancer.ResultsOverexpression of Gasp1 led to an increase in muscle mass observed not before day 15 of postnatal life. The surGasp1 transgenic mice did not display any other gross abnormality. Histological and morphometric analysis of surGasp1 rectus femoris muscles revealed an increase in myofiber size without a corresponding increase in myofiber number. Fiber-type distribution was unaltered. Interestingly, we do not detect a change in total fat mass and lean mass. These results differ from those for myostatin knockout mice, transgenic mice overexpressing the myostatin propeptide or follistatin which exhibit both muscle hypertrophy and hyperplasia, and show minimal fat deposition.ConclusionsAltogether, our data give new insight into the in vivo functions of Gasp1. As an extracellular regulatory factor in the myostatin signalling pathway, additional studies on GASP1 and its homolog GASP2 are required to elucidate the crosstalk between the different intrinsic inhibitors of the myostatin.

Murine GASP-1 N-Glycosylation is not Essential for its Activity on C2C12 Myogenic Cells but Alters its Secretion

Background/Aims: Growth and differentiation factor-associated serum protein-1 (GASP-1) is a secreted protein known to be capable of binding and inhibiting the activity of several TGF-beta family members, including myostatin. The present study was designed to characterize murine GASP-1 post-translational modifications and to determine their influence on the biological activity of GASP-1. Methods: We describe herein the site-directed mutagenesis of single N-glycosylation sites and combinations of them in 4 mutants of murine GASP-1. Results: In vitro and in vivo analysis revealed that GASP-1 is a glycoprotein containing 2 N-glycans and several mucin-type O-glycans. Treatments by the recombinant murine GASP-1 protein enhance C2C12 proliferation and differentiation by inhibition of the myostatin pathway. The loss of N-glycans leads to a decrease in protein secretion rate but does not affect its ability to activate myogenesis. Conclusion: Analysis of structure-function relationships of murine GASP-1 provides insights into the involvement of the carbohydrate moiety of mGASP-1 on its biological activity.

GASP/WFIKKN Proteins: Evolutionary Aspects of Their Functions

Growth and differentiation factor Associated Serum Protein (GASP) 1 and 2 are proteins known to be involved in the control of myostatin activity at least in vitro. Most deuterostome GASPs share a modular organization including WAP, follistatin/kazal, IGc2, two kunitz, and NTR domains. Based on an exon shuffling model, we performed independent phylogenetic analyses on these modules and assessed that papilin is probably a sister sequence to GASP with a divergence date estimated from the last common ancestor to bilateria. The final organization was acquired by the addition of the FS domain in early deuterostomes. Our study revealed that Gasp genes diverged during the first round of genome duplication in early vertebrates. By evaluating the substitution rate at different sites on the proteins, we showed a better conservation of the follistatin/kazal domain of GASP1 than GASP2 in mammals, suggesting a stronger interaction with myostatin. We also observed a progressive increase in the conservation of follistatin and kunitz domains from the ancestor of Ciona to early vertebrates. In situ hybridization performed on mouse embryos showed a weak Gasp1 expression in the formed somites at 10.5 dpc and in limb buds from embryonic E10.0 to E12.5. Similar results were obtained for zebrafish embryos. We propose a synthetic view showing possible interactions between GASP1 and myostatin and highlighting the role of the second kunitz domain in preventing myostatin proteolysis.

Construction of a large collection of small genome variations in French dairy and beef breeds using whole-genome sequences.

BackgroundIn recent years, several bovine genome sequencing projects were carried out with the aim of developing genomic tools to improve dairy and beef production efficiency and sustainability.ResultsIn this study, we describe the first French cattle genome variation dataset obtained by sequencing 274 whole genomes representing several major dairy and beef breeds. This dataset contains over 28 million single nucleotide polymorphisms (SNPs) and small insertions and deletions. Comparisons between sequencing results and SNP array genotypes revealed a very high genotype concordance rate, which indicates the good quality of our data.ConclusionsTo our knowledge, this is the first large-scale catalog of small genomic variations in French dairy and beef cattle. This resource will contribute to the study of gene functions and population structure and also help to improve traits through genotype-guided selection.

A sensitised mutagenesis screen in the mouse to explore the bovine genome: study of muscle characteristics.

Meat yield and quality are closely related to muscle development. The muscle characteristics mainly take place during embryonic and postnatal phases. Thus, genetic control of muscle development in early stages represents a significant stake to improve product quality and production efficiency. In bovine, several programmes have been developed to detect quantitative trait loci (QTL) affecting growth, carcass composition or meat quality traits. Such strategy is incontestably very powerful yet extremely cumbersome and costly when dealing with large animals such as ruminants. Furthermore, the fine mapping of the QTL remains a real challenge. Here, we proposed an alternative approach based on chemical mutagenesis in the mouse combined with comparative genomics to identify regions or genes controlling muscle development in cattle. At present, we isolated seven independent mouse lines of high interest. Two lines exhibit a hypermuscular phenotype, and the other five show various skeletomuscular phenotypes. Detailed characterisation of these mouse mutants will give crucial input for the identification and the mapping of genes that control muscular development. Our strategy will provide the opportunity to understand the function and control of genes involved in improvement of animal physiology.

An siRNA-based screen in C2C12 myoblasts identifies novel genes involved in myogenic differentiation

&NA; Myogenesis is a highly regulated multi‐step process involving myoblast proliferation and differentiation. Although studies over the last decades have identified several factors governing these distinct major phases, many of them are not yet known. In order to identify novel genes, we took advantage of the C2C12 myoblastic line to establish a functional siRNA screen combined with quantitative‐imaging analysis of a large amount of differentiated myoblasts. We knocked down 100 preselected mouse genes without a previously characterized role in muscle. Using image analysis, we tracked gene‐silencing phenotypes by quantitative assessment of cellular density, myotube quantity, myotube morphology and fusion index. Our results have revealed six genes involved in both stages of C2C12 myogenesis and 13 genes specific to the differentiation stage. These findings prove that our RNAi‐based screen could be an efficient tool to detect clear and subtle phenotypes allowing the identification of new myogenic regulators in mammals. HighlightsA new optimized protocol to perform a functional siRNA based screen.100 preselected genes knocked‐down in C2C12 myoblastic cell line.19 candidates genes identified as novel myogenic actors.

Genetic homogeneity of North-African goats

North Africa represents a rich and early reservoir of goat genetic diversity, from which the main African breeds have been derived. In this study, the genetic diversity of four indigenous Algerian goat breeds (i.e., Arabia, Makatia, M’Zabite and Kabyle, with n = 12 for each breed) has been investigated for the first time by genome-wide SNP genotyping; moreover in a broader context, genetic structuration of Algerian and Moroccan goats was explored (via FST, MDS, STRUCTURE, FineSTRUCTURE, BAPS, sPCA and DAPC analyses). At national level, the study revealed high level of genetic diversity and a significant phenomenon of admixture affecting all the Algerian breeds. At broader scale, clear global genetic homogeneity appeared considering both Algerian and Moroccan stocks. Indeed, genetic structuration was almost nonexistent among Arabia (from Algeria), Draa, Black and Nord (from Morocco), while the ancestral Kabyle and M’Zabite breeds, reared by Berber peoples, showed genetic distinctness. The study highlighted the threat to the Maghrebin stock, probably induced by unsupervised cross-breeding practices which have intensified in recent centuries. Moreover, it underlined the necessity to deepen our understanding of the genetic resources represented by the resilient North-African goat stock.

CNV-LDC: An Optimized CNV Detection Method for Low Depth of Coverage Data.

Recent improvements in technologies showed much greater variance of our genome than we thought. A part of this variance is due to submicroscopic chromosomal deletions/duplications called Copy Number Variations (CNVs). For some of these CNVs, it was clearly demonstrated that they play an important role in disease susceptibility, including complex diseases and Mendelian diseases. Last advances in next-generation sequencing have made fast progress in analyzing data for CNVs, in so far as they promise to improve the sensitivity in detection. This has led to the development of several new bioinformatics approaches and algorithms for detecting CNVs from this data for the four common methods: Assembly Based, Split Read, Read-Paired mapping, and Read Depth. Here we focus on the RD method that is able to detect the exact number of CNVs in comparison with the other methods. We propose an alternative method for detecting CNVs from short sequencing reads, CNV-LDC (Copy Number Variation-Low Depth of Coverage), that complements the existing method named CNV-TV (Copy Number Variation-Total Variation). We optimize the signal modeling and threshold step to lift the performance in low depth of coverage. Results of this new approach have been compared to various recent methods on different simulated data using small and large CNVs.

Assessing patterns of genetic admixture between sheep breeds: Case study in Algeria

Abstract In developing countries, cross‐breeding between local breeds and indigene or exotic breeds represents one of the main threats to the livestock diversity, leading to genetic dilution and loss of unique allelic combination underlying essential local adaptive traits. In this study, two Algerian sheep breeds, known to be highly admixed, were considered as a case study, to demonstrate how combination of different methodologies coupled with the use of specific softwares can be efficient to assess the spatial structuration of a hybrid zone, even in a case of extreme admixture. A fine sampling covering distribution areas of both breeds was implemented in order to study the admixture area and adjacent zones from a phenotypic (i.e., 19 quantitative traits were considered) and a genetic point of view (i.e., 21 microsatellites markers were used). Both approaches gave concordant patterns, highlighting areas with sheep most differentiated (or less admixed) for each breed. In detail, the region of Biskra appeared as the most preserved for the Ouled‐Djellal breed and the northwest of Laghouat was identified as the most preserved area for the Rembi breed. The approach proposed in the study offers a low‐cost solution to identify the most representative flocks of a breed, allowing the implementation of efficient conservation plans.