Tex19 and Sectm1 concordant molecular phylogenies support co-evolution of both eutherian-specific genes

International audienceBackground: Transposable elements (TE) have attracted much attention since they shape the genome and contribute to species evolution. Organisms have evolved mechanisms to control TE activity. Testis expressed 19 (Tex19) represses TE expression in mouse testis and placenta. In the human and mouse genomes, Tex19 and Secreted and transmembrane 1 (Sectm1) are neighbors but are not homologs. Sectm1 is involved in immunity and its molecular phylogeny is unknown. Methods: Using multiple alignments of complete protein sequences (MACS), we inferred Tex19 and Sectm1 molecular phylogenies. Protein conserved regions were identified and folds were predicted. Finally, expression patterns were studied across tissues and species using RNA-seq public data and RT-PCR. Results: We present 2 high quality alignments of 58 Tex19 and 58 Sectm1 protein sequences from 48 organisms. First, both genes are eutherian-specific, i.e., exclusively present in mammals except monotremes (platypus) and marsupials. Second, Tex19 and Sectm1 have both duplicated in Sciurognathi and Bovidae while they have remained as single copy genes in all further placental mammals. Phylogenetic concordance between both genes was significant (p-value < 0.05) and supported co-evolution and functional relationship. At the protein level, Tex19 exhibits 3 conserved regions and 4 invariant cysteines. In particular, a CXXC motif is present in the N-terminal conserved region. Sectm1 exhibits 2 invariant cysteines and an Ig-like domain. Strikingly, Tex19 C-terminal conserved region was lost in Haplorrhini primates while a Sectm1 C-terminal extra domain was acquired. Finally, we have determined that Tex19 and Sectm1 expression levels anti-correlate across the testis of several primates (ρ = −0.72) which supports anti-regulation. Conclusions: Tex19 and Sectm1 co-evolution and anti-regulated expressions support a strong functional relationship between both genes. Since Tex19 operates a control on TE and Sectm1 plays a role in immunity, Tex19 might suppress an immune response directed against cells that show TE activity in eutherian reproductive tissues

Extended passaging increases the efficiency of neural differentiation from induced pluripotent stem cells

<p>Abstract</p> <p>Background</p> <p>The use of induced pluripotent stem cells (iPSCs) for the functional replacement of damaged neurons and <it>in vitro </it>disease modeling is of great clinical relevance. Unfortunately, the capacity of iPSC lines to differentiate into neurons is highly variable, prompting the need for a reliable means of assessing the differentiation capacity of newly derived iPSC cell lines. Extended passaging is emerging as a method of ensuring faithful reprogramming. We adapted an established and efficient embryonic stem cell (ESC) neural induction protocol to test whether iPSCs (1) have the competence to give rise to functional neurons with similar efficiency as ESCs and (2) whether the extent of neural differentiation could be altered or enhanced by increased passaging.</p> <p>Results</p> <p>Our gene expression and morphological analyses revealed that neural conversion was temporally delayed in iPSC lines and some iPSC lines did not properly form embryoid bodies during the first stage of differentiation. Notably, these deficits were corrected by continual passaging in an iPSC clone. iPSCs with greater than 20 passages (late-passage iPSCs) expressed higher expression levels of pluripotency markers and formed larger embryoid bodies than iPSCs with fewer than 10 passages (early-passage iPSCs). Moreover, late-passage iPSCs started to express neural marker genes sooner than early-passage iPSCs after the initiation of neural induction. Furthermore, late-passage iPSC-derived neurons exhibited notably greater excitability and larger voltage-gated currents than early-passage iPSC-derived neurons, although these cells were morphologically indistinguishable.</p> <p>Conclusions</p> <p>These findings strongly suggest that the efficiency neuronal conversion depends on the complete reprogramming of iPSCs via extensive passaging.</p

Highly dynamic and sex-specific expression of microRNAs during early ES cell differentiation.

International audienceEmbryonic stem (ES) cells are pluripotent cells derived from the inner cell mass of the mammalian blastocyst. Cellular differentiation entails loss of pluripotency and gain of lineage-specific characteristics. However, the molecular controls that govern the differentiation process remain poorly understood. We have characterized small RNA expression profiles in differentiating ES cells as a model for early mammalian development. High-throughput 454 pyro-sequencing was performed on 19-30 nt RNAs isolated from undifferentiated male and female ES cells, as well as day 2 and 5 differentiating derivatives. A discrete subset of microRNAs (miRNAs) largely dominated the small RNA repertoire, and the dynamics of their accumulation could be readily used to discriminate pluripotency from early differentiation events. Unsupervised partitioning around meloids (PAM) analysis revealed that differentiating ES cell miRNAs can be divided into three expression clusters with highly contrasted accumulation patterns. PAM analysis afforded an unprecedented level of definition in the temporal fluctuations of individual members of several miRNA genomic clusters. Notably, this unravelled highly complex post-transcriptional regulations of the key pluripotency miR-290 locus, and helped identify miR-293 as a clear outlier within this cluster. Accordingly, the miR-293 seed sequence and its predicted cellular targets differed drastically from those of the other abundant cluster members, suggesting that previous conclusions drawn from whole miR-290 over-expression need to be reconsidered. Our analysis in ES cells also uncovered a striking male-specific enrichment of the miR-302 family, which share the same seed sequence with most miR-290 family members. Accordingly, a miR-302 representative was strongly enriched in embryonic germ cells derived from primordial germ cells of male but not female mouse embryos. Identifying the chromatin remodelling and E2F-dependent transcription repressors Ari4a and Arid4b as additional targets of miR-302 and miR-290 supports and possibly expands a model integrating possible overlapping functions of the two miRNA families in mouse cell totipotency during early development. This study demonstrates that small RNA sampling throughout early ES cell differentiation enables the definition of statistically significant expression patterns for most cellular miRNAs. We have further shown that the transience of some of these miRNA patterns provides highly discriminative markers of particular ES cell states during their differentiation, an approach that might be broadly applicable to the study of early mammalian development

Cell Transplant

Human induced pluripotent stem cells (hiPSCs) are a most appealing source for cell replacement therapy in acute brain lesions. We evaluated the potential of hiPSC therapy in stroke by transplanting hiPSC-derived neural progenitor cells (NPCs) into the postischemic striatum. Grafts received host tyrosine hydroxylase-positive afferents and contained developing interneurons and homotopic GABAergic medium spiny neurons that, with time, sent axons to the host substantia nigra. Grafting reversed stroke-induced somatosensory and motor deficits. Grafting also protected the host substantia nigra from the atrophy that follows disruption of reciprocal striatonigral connections. Graft innervation by tyrosine hydoxylase fibers, substantia nigra protection, and somatosensory functional recovery were early events, temporally dissociated from the slow maturation of GABAergic neurons in the grafts and innervation of substantia nigra. This suggests that grafted hiPSC-NPCs initially exert trophic effects on host brain structures, which precede integration and potential pathway reconstruction. We believe that transplantation of NPCs derived from hiPSCs can provide useful interventions to limit the functional consequences of stroke through both neuroprotective effects and reconstruction of impaired pathways

Current issues in medically assisted reproduction and genetics in Europe: research, clinical practice, ethics, legal issues and policy. European Society of Human Genetics and European Society of Human Reproduction and Embryology.

In March 2005, a group of experts from the European Society of Human Genetics and European Society of Human Reproduction and Embryology met to discuss the interface between genetics and assisted reproductive technology (ART), and published an extended background paper, recommendations and two Editorials. Seven years later, in March 2012, a follow-up interdisciplinary workshop was held, involving representatives of both professional societies, including experts from the European Union Eurogentest2 Coordination Action Project. The main goal of this meeting was to discuss developments at the interface between clinical genetics and ARTs. As more genetic causes of reproductive failure are now recognised and an increasing number of patients undergo testing of their genome before conception, either in regular health care or in the context of direct-to-consumer testing, the need for genetic counselling and preimplantation genetic diagnosis (PGD) may increase. Preimplantation genetic screening (PGS) thus far does not have evidence from randomised clinical trials to substantiate that the technique is both effective and efficient. Whole-genome sequencing may create greater challenges both in the technological and interpretational domains, and requires further reflection about the ethics of genetic testing in ART and PGD/PGS. Diagnostic laboratories should be reporting their results according to internationally accepted accreditation standards (International Standards Organisation - ISO 15189). Further studies are needed in order to address issues related to the impact of ART on epigenetic reprogramming of the early embryo. The legal landscape regarding assisted reproduction is evolving but still remains very heterogeneous and often contradictory. The lack of legal harmonisation and uneven access to infertility treatment and PGD/PGS fosters considerable cross-border reproductive care in Europe and beyond. The aim of this paper is to complement previous publications and provide an update of selected topics that have evolved since 2005

Caractérisation et étude fonctionnelle de Tex19.1 dans l embryon précoce et la pluripotence des cellules souches embryonnaires

La pluripotence permet à l embryon de former tous les tissus des trois feuillets embryonnaires. Après la gastrulation, seules les cellules germinales primordiales (PGC) maintiennent un caractère pluripotent, indispensable à la génération d un nouvel embryon à la génération suivante. Les PGC ont donc des caractéristiques similaires aux cellules souches de l embryon précoce et aux cellules souches embryonnaires (ESC), comme l expression des gènes de pluripotence. Dans le but d identifier un nouveau marqueur de pluripotence, nous avons cherché un gène spécifique des cellules germinales puis avons étudié sa fonction dans les cellules souches pluripotentes. Nous avons choisi d étudier Tex19.1 pour son profil d expression restreint aux gonades. Nous avons montré qu il est exprimé spécifiquement dans les cellules pluripotentes de l embryon et les PGC ainsi que dans le placenta. Son expression est transmise aux cellules souches pluripotentes in vitro comme les ESC. De plus, celle-ci semble requise pour l état pluripotent puisqu elle est induite lorsque des fibroblastes sont reprogrammés en cellules souches à pluripotence induite (iPSC). Pourtant, des études de gain et de perte de fonction dans les ESC montrent que Tex19.1 n est pas indispensable à l établissement ou au maintien de la pluripotence. Les animaux mutants pour Tex19.1 montrent un défaut lors de la spermatogenèse entrainant la stérilité des males. De plus, la moitié d entre eux meure à un stade et pour une cause inconnus à l heure actuelle. Tex19.1 pourrait jouer un rôle dans la différenciation des ESC en cellules germinales, et dans la reprogrammation épigénétique des PGC in vivo.Pluripotency allows the embryo to form all tissues of the three germ layers. After gastrulation, the only population which remain pluripotent are the primordial germ cells (PGC), that are able to give rise to a whole embryo at the next generation. Then, PGC, pluripotent cells of the early embryo and embryonic stem cells (ESC) have common properties, such as the expression of key pluripotency genes. To identify new pluripotency markers, we lokked for a germ cell specific gene and then analysed its function in pluripotent stem cells. We studied Tex19.1 for its specific expression in the gonads.We showed that it was specifically expressed in pluripotent cells of the embryo and in PGC and placenta. Its expression is transmitted to the pluripotent cell line in vitro, like ESC. Moreover, its expression seems to be required for the pluripotent state since it s induced when fibroblasts are reprogrammed into induced pluripotent stem cells (iPSC). However, gain- and loss-of-function studies in ESC showed that Tex19.1 is dispensable for pluripotency establishment and maintenance. Tex19.1 mutant animals have a spermatogenesis defect, causing sterility of the males. Moreover, half of the mutant animals are dying, at a stage and for a cause that remain to be determined. Tex19.1 may play a role in ESC differentiation into germ cells, and in PGC epigenetic reprogramming in vivo.STRASBOURG-Sc. et Techniques (674822102) / SudocSudocFranceF

Nouvelles techniques non invasives de diagnostic prénatal par analyse des cellules foetales et ADN libre foetal circulant dans le sang maternel

STRASBOURG ILLKIRCH-Pharmacie (672182101) / SudocSudocFranceF

Génétique de l'infertilité masculine

STRASBOURG ILLKIRCH-Pharmacie (672182101) / SudocSudocFranceF

Étude du protéome des cellules souches embryonnaires et des cellules germinales embryonnaires chez la souris à la recherche de nouveaux marqueurs de pluripotence

Les cellules souches (ES) et germinales embryonnaires (EG) sont les 2 principales sources de cellules pluripotentes connues chez les mammifères, et sont un modèle d étude intéressant pour une meilleure compréhension des mécanismes de la pluripotence. Leurs propriétés particulières en font des candidats attractifs pour leur utilisation en thérapie de remplacement cellulaire. Afin d étudier les voies de signalisation impliquées dans la pluripotence, une étude protéomique a été réalisée à partir des cellules ES et EG de souris. Une stratégie de soustraction des protéines contaminantes de l environnement de culture a été appliquée pour l étude du protéome spécifique de ces cellules. Cette approche a permis l identification de deux isoformes (acétylée et non-acétylée) d un marqueur de pluripotence connu, nommé DPPA5. Enfin, cette stratégie de soustraction couplée à un sous-fractionnement nucléaire a permis l identification de PRMT7, un nouveau marqueur potentiel de la pluripotence.Embryonic stem cells (ESCs) and embryonic germ cells (EGCs) are the major pluripotent mammalian cells currently known and provide exciting models for understanding the underlying mechanisms that make a cell pluripotent. Furthermore, their particular properties make ESCs and EGCs very attractive candidates for cell replacement therapy. In order to investigate signaling pathways potentially involved in pluripotency, proteomic analyses have been performed on mouse ESC and EGC proteins. A subtractive strategy (subtracting culture environment contaminating proteins) was applied for the study of the specific proteome of these cells. This approach led to the identification of two isoforms (with and without N-terminal acetylation) of a known pluripotency marker, namely DPPA5. Furthermore, we demonstrated the efficiency of our subtracting strategy, in association with a nuclear subfractionation by the identification of a new protein (PRMT7) behaving as proteins involved in pluripotency.STRASBOURG-Sc. et Techniques (674822102) / SudocSudocFranceF

Human genetics of male infertility

Le génotypage d une famille jordanienne consanguine constituée de 5 frères globozoospermiques et de 3 frères fertiles sur puce Affymetrix, a permis d identifier un nouveau gène responsable de la globozoospermie situé dans un intervalle de 6.4Mb en 12q14.2. Au regard de son expression prédominante dans le testicule et l implication de son orthologue, chez C. elegans, dans la polarisation cellulaire, le gène DPY19L2 est un gène candidat parfait. Le gène, codant pour une protéine transmembranaire, est flanqué par deux séquences répétées (LCRs) qui partagent 96,5% d identité. Dans une première étude, une délétion de 200Kb englobant l ensemble du gène a été mise en évidence chez les 4 frères infertiles de cette famille jordanienne ainsi que chez 3 autres patients non apparentés. Nous avons ensuite recruté une plus grande cohorte de 54 patients. Parmi ces patients, 20 sont homozygotes pour la délétion de DPY19L2 et 7 sont hétérozygotes composites associant la délétion hétérozygote et une mutation ponctuelle. En outre, nous avons identifié, 4 patients avec des mutations ponctuelles homozygotes. Par conséquent, la fréquence d implication de DPY19L2 s élève à 66.7%. En tout, 9 points de cassures, regroupés en deux hotspots au sein des LCRs, ont pu être mis en évidence. Ceci confirme que le mécanisme sous-jacent de la délétion est une recombinaison homologue non allélique (NAHR) entre les LCRs. En conclusion, nous confirmons que DPY19L2 est le principal gène de la globozoospermie et nous élargissons le spectre des mutations possible dans ce gène.Performing a genome wide scan by SNP microarray on a Jordanian consanguineous family where five brothers were diagnosed with complete globozoospermia, we show in a first study that the four out of five analysed infertile brothers carried a homozygous deletion of 200 kb on chromosome 12 encompassing only DPY19L2. The gene encodes for a transmembrane protein and is surrounded by two low copy repeats (LCRs). Very similar deletions were found in three additional unrelated patients. Later, we have pursued our patient screen by recruiting a largest cohort of patients. Out of a total of 54 patients analysed, 36 (66.7%) showed a mutation in DPY19L2. Out of 36 mutated patients, 20 are homozygous deleted, 7 heterozygous composite and 4 showed a homozygous point mutation. We characterized a total of nine breakpoints that clustered in two recombination hotspots, both containing direct repeat elements. These findings confirm that the deletion is due to a nonallelic homologous recombination (NAHR) between the two LCRs. Thus, Globozoospermia can be considered as a new genomic disorder. This study confirms that DPY19L2 is the major gene responsible for globozoospermia and enlarges the spectrum of possible mutations in the gene.STRASBOURG-Bib.electronique 063 (674829902) / SudocSudocFranceF