Cédric Cabau

The rainbow trout genome provides novel insights into evolution after whole-genome duplication in vertebrates

Vertebrate evolution has been shaped by several rounds of whole-genome duplications (WGDs) that are often suggested to be associated with adaptive radiations and evolutionary innovations. Due to an additional round of WGD, the rainbow trout genome offers a unique opportunity to investigate the early evolutionary fate of a duplicated vertebrate genome. Here we show that after 100 million years of evolution the two ancestral subgenomes have remained extremely collinear, despite the loss of half of the duplicated protein-coding genes, mostly through pseudogenization. In striking contrast is the fate of miRNA genes that have almost all been retained as duplicated copies. The slow and stepwise rediploidization process characterized here challenges the current hypothesis that WGD is followed by massive and rapid genomic reorganizations and gene deletions.

IMpRH server: an RH mapping server available on the Web.

SUMMARY The INRA-Minnesota Porcine Radiation Hybrid (IMpRH) Server provides both a mapping tool (IMpRH mapping tool) and a database (IMpRH database) of officially submitted results. The mapping tool permits the mapping of a new marker relatively to markers previously mapped on the IMpRH panel. The IMpRH database is the official database for submission of new results and queries. The database not only permits the sharing of public data but also semi-private and private data.

Design and Characterization of a 52K SNP Chip for Goats

The success of Genome Wide Association Studies in the discovery of sequence variation linked to complex traits in humans has increased interest in high throughput SNP genotyping assays in livestock species. Primary goals are QTL detection and genomic selection. The purpose here was design of a 50–60,000 SNP chip for goats. The success of a moderate density SNP assay depends on reliable bioinformatic SNP detection procedures, the technological success rate of the SNP design, even spacing of SNPs on the genome and selection of Minor Allele Frequencies (MAF) suitable to use in diverse breeds. Through the federation of three SNP discovery projects consolidated as the International Goat Genome Consortium, we have identified approximately twelve million high quality SNP variants in the goat genome stored in a database together with their biological and technical characteristics. These SNPs were identified within and between six breeds (meat, milk and mixed): Alpine, Boer, Creole, Katjang, Saanen and Savanna, comprising a total of 97 animals. Whole genome and Reduced Representation Library sequences were aligned on >10 kb scaffolds of the de novo goat genome assembly. The 60,000 selected SNPs, evenly spaced on the goat genome, were submitted for oligo manufacturing (Illumina, Inc) and published in dbSNP along with flanking sequences and map position on goat assemblies (i.e. scaffolds and pseudo-chromosomes), sheep genome V2 and cattle UMD3.1 assembly. Ten breeds were then used to validate the SNP content and 52,295 loci could be successfully genotyped and used to generate a final cluster file. The combined strategy of using mainly whole genome Next Generation Sequencing and mapping on a contig genome assembly, complemented with Illumina design tools proved to be efficient in producing this GoatSNP50 chip. Advances in use of molecular markers are expected to accelerate goat genomic studies in coming years.

Gene expression profiling to identify eggshell proteins involved in physical defense of the chicken egg

BackgroundAs uricoletic animals, chickens produce cleidoic eggs, which are self-contained bacteria-resistant biological packages for extra-uterine development of the chick embryo. The eggshell constitutes a natural physical barrier against bacterial penetration if it forms correctly and remains intact. The eggshells remarkable mechanical properties are due to interactions among mineral components and the organic matrix proteins. The purpose of our study was to identify novel eggshell proteins by examining the transcriptome of the uterus during calcification of the eggshell. An extensive bioinformatic analysis on genes over-expressed in the uterus allowed us to identify novel eggshell proteins that contribute to the eggs natural defenses.ResultsOur 14 K Del-Mar Chicken Integrated Systems microarray was used for transcriptional profiling in the hens uterus during eggshell deposition. A total of 605 transcripts were over-expressed in the uterus compared with the magnum or white isthmus across a wide range of abundance (1.1- to 79.4-fold difference). The 605 highly-expressed uterine transcripts correspond to 469 unique genes, which encode 437 different proteins. Gene Ontology (GO) analysis was used for interpretation of protein function. The most over-represented GO terms are related to genes encoding ion transport proteins, which provide eggshell mineral precursors. Signal peptide sequence was found for 54 putative proteins secreted by the uterus during eggshell formation. Many functional proteins are involved in calcium binding or biomineralization--prerequisites for interacting with the mineral phase during eggshell fabrication. While another large group of proteins could be involved in proper folding of the eggshell matrix. Many secreted uterine proteins possess antibacterial properties, which would protect the egg against microbial invasion. A final group includes proteases and protease inhibitors that regulate protein activity in the acellular uterine fluid where eggshell formation takes place.ConclusionsOur original study provides the first detailed description of the chicken uterus transcriptome during formation of the eggshell. We have discovered a cache of about 600 functional genes and identified a large number of encoded proteins secreted into uterine fluid for fabrication of the eggshell and chemical protection of the egg. Some of these uterine genes could prove useful as biological markers for genetic improvement of phenotypic traits (i.e., egg and eggshell quality).

Kinetics of gene expression and signaling in bovine cumulus cells throughout IVM in different mediums in relation to oocyte developmental competence, cumulus apoptosis and progesterone secretion

In vitro maturation of oocytes is a crucial step in assisted reproductive technologies in cattle; however, the molecular mechanisms of cumulus contribution to oocyte developmental potential require more investigation. Based on transcriptomic data, we studied by using real-time RT-PCR and western blot in bovine cumulus cells, the kinetics of expression of several candidate genes involved in oxidative stress response, apoptosis, steroid metabolism and signal transmission throughout IVM. Phosphorylations of the components of the main signaling pathways were also analyzed. In addition, IVM was performed in different maturation mediums which influenced the cumulus apoptosis, progesterone secretion and oocyte developmental competence. Glutathione-S-transferase A1 (GSTA1) transcript and protein abundance significantly decreased throughout IVM progression. Similarly, transcript levels of FSH receptor and aromatase (CYP19A1) and protein levels of three steroidogenic enzymes (steroidogenic acute regulatory protein, cytochrome P450scc and 3-beta-hydroxysteroid dehydrogenase) decreased along with progression of maturation and especially since 10 hours of IVM. Expression of progesterone receptor (PGR) and clusterin (CLU) mRNA and phosphorylations of protein kinases AKT, MAPK P38 and SMAD2 were particularly increased at 10 hours of IVM. This expression pattern supposed the role of these factors during oocyte metaphase-I check point of meiosis. Levels of CLU, GSTA1 and FSHR transcripts were higher in 199 basic hormone-free medium as compared to the medium 199EM, enriched in gonadotropins and growth factors, in which we recorded the higher developmental rate and progesterone secretion. Higher phosphorylation levels of SMAD2, AKT and MAP kinase JNK1, but not of MAP kinases ERK1/ERK2 or P38, was positively correlated with oocyte developmental competence and progesterone secretion and negatively correlated with cumulus apoptosis rate. These factors and signaling pathways in cumulus cells are potentially involved in controlling different stages of oocyte nuclear maturation and acquirement of its developmental potential.

Contribution to high-resolution mapping in pigs with 101 type I markers and progress in comparative map between humans and pigs.

In the frame of the European program GenetPig, we localized on the Pig map 105 coding sequences (type I markers) from different origins, using INRA-University of Minnesota porcine Radiation Hybrid Panel (IMpRH, 101 markers) and somatic cell hybrid panel (SCHP, 93 markers, of which only four were not also mapped using IMpRH). Thus, we contributed to the improvement of the porcine high-resolution map, and we complemented the integration between the RH and cytogenetic maps. IMpRH tools allowed us to map 101 new markers relatively to reference markers of the first generation radiation hybrid map. Ninety out of 101 markers are linked to an already mapped marker with a LOD score greater than 4.8. Seventy-eight markers were informative for comparative mapping. Comparison of marker positions on the RH map with those obtained on the cytogenetic map or those expected by Human-Pig comparative map data suggested to us to be cautious with markers linked with a LOD lower than 6. These results allowed us to specify chromosomal fragments well conserved between humans and pigs and also to suggest new correspondences (Sscr1-Hsap3, Sscr9-Hsap9, Sscr13-Hsap11, Sscr15-Hsap6) confirmed by FISH on pig chromosomes. We examined in more detail the comparative map between Hsap12 and Sscr5 considering gene order, which suggests that rearrangements have occurred within the conserved synteny.

Strengthening insights into host responses to mastitis infection in ruminants by combining heterogeneous microarray data sources

BackgroundGene expression profiling studies of mastitis in ruminants have provided key but fragmented knowledge for the understanding of the disease. A systematic combination of different expression profiling studies via meta-analysis techniques has the potential to test the extensibility of conclusions based on single studies. Using the program Pointillist, we performed meta-analysis of transcription-profiling data from six independent studies of infections with mammary gland pathogens, including samples from cattle challenged in vivo with S. aureus, E. coli, and S. uberis, samples from goats challenged in vivo with S. aureus, as well as cattle macrophages and ovine dendritic cells infected in vitro with S. aureus. We combined different time points from those studies, testing different responses to mastitis infection: overall (common signature), early stage, late stage, and cattle-specific.ResultsIngenuity Pathway Analysis of affected genes showed that the four meta-analysis combinations share biological functions and pathways (e.g. protein ubiquitination and polyamine regulation) which are intrinsic to the general disease response. In the overall response, pathways related to immune response and inflammation, as well as biological functions related to lipid metabolism were altered. This latter observation is consistent with the milk fat content depression commonly observed during mastitis infection. Complementarities between early and late stage responses were found, with a prominence of metabolic and stress signals in the early stage and of the immune response related to the lipid metabolism in the late stage; both mechanisms apparently modulated by few genes, including XBP1 and SREBF1.The cattle-specific response was characterized by alteration of the immune response and by modification of lipid metabolism. Comparison of E. coli and S. aureus infections in cattle in vivo revealed that affected genes showing opposite regulation had the same altered biological functions and provided evidence that E. coli caused a stronger host response.ConclusionsThis meta-analysis approach reinforces previous findings but also reveals several novel themes, including the involvement of genes, biological functions, and pathways that were not identified in individual studies. As such, it provides an interesting proof of principle for future studies combining information from diverse heterogeneous sources.

An overview of gene expression dynamics during early ovarian folliculogenesis: specificity of follicular compartments and bi-directional dialog

BackgroundSuccessful early folliculogenesis is crucial for female reproductive function. It requires appropriate gene specific expression of the different types of ovarian cells at different developmental stages. To date, most gene expression studies on the ovary were conducted in rodents and did not distinguish the type of cell. In mono-ovulating species, few studies have addressed gene expression profiles and mainly concerned human oocytes.ResultsWe used a laser capture microdissection method combined with RNA-seq technology to explore the transcriptome in oocytes and granulosa cells (GCs) during development of the sheep ovarian follicle. We first documented the expression profile of 15 349 genes, then focused on the 5 129 genes showing differential expression between oocytes and GCs. Enriched functional categories such as oocyte meiotic arrest and GC steroid synthesis reflect two distinct cell fates. We identified the implication of GC signal transduction pathways such as SHH, WNT and RHO GTPase. In addition, signaling pathways (VEGF, NOTCH, IGF1, etc.) and GC transzonal projections suggest the existence of complex cell-cell interactions. Finally, we highlighted several transcription regulators and specifically expressed genes that likely play an important role in early folliculogenesis.ConclusionsTo our knowledge, this is the first comprehensive exploration of transcriptomes derived from in vivo oocytes and GCs at key stages in early follicular development in sheep. Collectively, our data advance our understanding of early folliculogenesis in mono-ovulating species and will be a valuable resource for unraveling human ovarian dysfunction such as premature ovarian failure (POF).

Induced pluripotent stem cells derived from rabbits exhibit some characteristics of naïve pluripotency

Summary Not much is known about the molecular and functional features of pluripotent stem cells (PSCs) in rabbits. To address this, we derived and characterized 2 types of rabbit PSCs from the same breed of New Zealand White rabbits: 4 lines of embryonic stem cells (rbESCs), and 3 lines of induced PSCs (rbiPSCs) that were obtained by reprogramming adult skin fibroblasts. All cell lines required fibroblast growth factor 2 for their growth and proliferation. All rbESC lines showed molecular and functional properties typically associated with primed pluripotency. The cell cycle of rbESCs had a prolonged G1 phase and a DNA damage checkpoint before entry into the S phase, which are the 2 features typically associated with the somatic cell cycle. In contrast, the rbiPSC lines exhibited some characteristics of naïve pluripotency, including resistance to single-cell dissociation by trypsin, robust activity of the distal enhancer of the mouse Oct4 gene, and expression of naïve pluripotency-specific genes, as defined in rodents. According to gene expression profiles, rbiPSCs were closer to the rabbit inner cell mass (ICM) than rbESCs. Furthermore, rbiPSCs were capable of colonizing the ICM after aggregation with morulas. Therefore, we propose that rbiPSCs self-renew in an intermediate state between naïve and primed pluripotency, which represents a key step toward the generation of bona fide naïve PSC lines in rabbits.

Revealing the dynamics of gene expression during embryonic genome activation and first differentiation in the rabbit embryo with a dedicated array screening

Early mammalian development is characterized by extensive changes in nuclear functions that result from epigenetic modifications of the newly formed embryonic genome. While the first embryonic cells are totipotent, this status spans only a few cell cycles. At the blastocyst stage, the embryo already contains differentiated trophectoderm cells and pluripotent inner cell mass cells. Concomitantly, the embryonic genome becomes progressively transcriptionally active. During this unique period of development, the gene expression pattern has been mainly characterized in the mouse, in which embryonic genome activation (EGA) spans a single cell cycle after abrupt epigenetic modifications. To further characterize this period, we chose to analyze it in the rabbit, in which, as in most mammals, EGA is more progressive and occurs closer to the first cell differentiation events. In this species, for which no transcriptomic arrays were available, we focused on genes expressed at EGA and first differentiation and established a 2,000-gene dedicated cDNA array. Screening this with pre-EGA, early post-EGA, and blastocyst embryos divided genes into seven clusters of expression according to their regulation during this period and revealed their dynamics of expression during EGA and first differentiation. Our results point to transient properties of embryo transcriptome at EGA, due not only to the transition between maternal and embryonic transcripts but also to the transient expression of a subset of embryonic genes whose functions remained largely uncharacterized. They also provide a first view of the functional consequences of the changes in gene expression program.