Publications by authors named "Laurent Journot"

38 Publications

RADSex: A computational workflow to study sex determination using restriction site-associated DNA sequencing data.

Mol Ecol Resour 2021 Jul 9;21(5):1715-1731. Epub 2021 Mar 9.

Physiological Chemistry, Biocenter, University of Wuerzburg, Wuerzburg, Germany.

The study of sex determination and sex chromosome organization in nonmodel species has long been technically challenging, but new sequencing methodologies now enable precise and high-throughput identification of sex-specific genomic sequences. In particular, restriction site-associated DNA sequencing (RAD-Seq) is being extensively applied to explore sex determination systems in many plant and animal species. However, software specifically designed to search for and visualize sex-biased markers using RAD-Seq data is lacking. Here, we present RADSex, a computational analysis workflow designed to study the genetic basis of sex determination using RAD-Seq data. RADSex is simple to use, requires few computational resources, makes no prior assumptions about the type of sex-determination system or structure of the sex locus, and offers convenient visualization through a dedicated R package. To demonstrate the functionality of RADSex, we re-analysed a published data set of Japanese medaka, Oryzias latipes, where we uncovered a previously unknown Y chromosome polymorphism. We then used RADSex to analyse new RAD-Seq data sets from 15 fish species spanning multiple taxonomic orders. We identified the sex determination system and sex-specific markers in six of these species, five of which had no known sex-markers prior to this study. We show that RADSex greatly facilitates the study of sex determination systems in nonmodel species thanks to its speed of analyses, low resource usage, ease of application and visualization options. Furthermore, our analysis of new data sets from 15 species provides new insights on sex determination in fish.
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http://dx.doi.org/10.1111/1755-0998.13360DOI Listing
July 2021

The rise and fall of the ancient northern pike master sex-determining gene.

Elife 2021 Jan 28;10. Epub 2021 Jan 28.

INRAE, Sigenae, Genotoul Bioinfo, Toulouse, France.

The understanding of the evolution of variable sex determination mechanisms across taxa requires comparative studies among closely related species. Following the fate of a known master sex-determining gene, we traced the evolution of sex determination in an entire teleost order (Esociformes). We discovered that the northern pike () master sex-determining gene originated from a 65 to 90 million-year-old gene duplication event and that it remained sex linked on undifferentiated sex chromosomes for at least 56 million years in multiple species. We identified several independent species- or population-specific sex determination transitions, including a recent loss of a Y chromosome. These findings highlight the diversity of evolutionary fates of master sex-determining genes and the importance of population demographic history in sex determination studies. We hypothesize that occasional sex reversals and genetic bottlenecks provide a non-adaptive explanation for sex determination transitions.
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http://dx.doi.org/10.7554/eLife.62858DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7870143PMC
January 2021

Sex chromosome and sex locus characterization in goldfish, Carassius auratus (Linnaeus, 1758).

BMC Genomics 2020 Aug 11;21(1):552. Epub 2020 Aug 11.

INRAE, LPGP, 35000, Rennes, France.

Background: Goldfish is an important model for various areas of research, including neural development and behavior and a species of significant importance in aquaculture, especially as an ornamental species. It has a male heterogametic (XX/XY) sex determination system that relies on both genetic and environmental factors, with high temperatures being able to produce female-to-male sex reversal. Little, however, is currently known on the molecular basis of genetic sex determination in this important cyprinid model. Here we used sequencing approaches to better characterize sex determination and sex-chromosomes in an experimental strain of goldfish.

Results: Our results confirmed that sex determination in goldfish is a mix of environmental and genetic factors and that its sex determination system is male heterogametic (XX/XY). Using reduced representation (RAD-seq) and whole genome (pool-seq) approaches, we characterized sex-linked polymorphisms and developed male specific genetic markers. These male specific markers were used to distinguish sex-reversed XX neomales from XY males and to demonstrate that XX female-to-male sex reversal could even occur at a relatively low rearing temperature (18 °C), for which sex reversal has been previously shown to be close to zero. We also characterized a relatively large non-recombining region (~ 11.7 Mb) on goldfish linkage group 22 (LG22) that contained a high-density of male-biased genetic polymorphisms. This large LG22 region harbors 373 genes, including a single candidate as a potential master sex gene, i.e., the anti-Mullerian hormone gene (amh). However, no sex-linked polymorphisms were detected in the coding DNA sequence of the goldfish amh gene.

Conclusions: These results show that our goldfish strain has a relatively large sex locus on LG22, which is likely the Y chromosome of this experimental population. The presence of a few XX males even at low temperature also suggests that other environmental factors in addition to temperature could trigger female-to-male sex reversal. Finally, we also developed sex-linked genetic markers, which will be important tools for future research on sex determination in our experimental goldfish population. However, additional work would be needed to explore whether this sex locus is conserved in other populations of goldfish.
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http://dx.doi.org/10.1186/s12864-020-06959-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7430817PMC
August 2020

The sterlet sturgeon genome sequence and the mechanisms of segmental rediploidization.

Nat Ecol Evol 2020 06 30;4(6):841-852. Epub 2020 Mar 30.

Developmental Biochemistry, Biocenter, University of Wuerzburg, Wuerzburg, Germany.

Sturgeons seem to be frozen in time. The archaic characteristics of this ancient fish lineage place it in a key phylogenetic position at the base of the ~30,000 modern teleost fish species. Moreover, sturgeons are notoriously polyploid, providing unique opportunities to investigate the evolution of polyploid genomes. We assembled a high-quality chromosome-level reference genome for the sterlet, Acipenser ruthenus. Our analysis revealed a very low protein evolution rate that is at least as slow as in other deep branches of the vertebrate tree, such as that of the coelacanth. We uncovered a whole-genome duplication that occurred in the Jurassic, early in the evolution of the entire sturgeon lineage. Following this polyploidization, the rediploidization of the genome included the loss of whole chromosomes in a segmental deduplication process. While known adaptive processes helped conserve a high degree of structural and functional tetraploidy over more than 180 million years, the reduction of redundancy of the polyploid genome seems to have been remarkably random.
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http://dx.doi.org/10.1038/s41559-020-1166-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7269910PMC
June 2020

Genome Sequence of the Euryhaline Javafish Medaka, : A Small Aquarium Fish Model for Studies on Adaptation to Salinity.

G3 (Bethesda) 2020 03 5;10(3):907-915. Epub 2020 Mar 5.

INRAE, UR 1037 Fish Physiology and Genomics, F-35000 Rennes, France, and

The genus consists of 35 medaka-fish species each exhibiting various ecological, morphological and physiological peculiarities and adaptations. Beyond of being a comprehensive phylogenetic group for studying intra-genus evolution of several traits like sex determination, behavior, morphology or adaptation through comparative genomic approaches, all medaka species share many advantages of experimental model organisms including small size and short generation time, transparent embryos and genome editing tools for reverse and forward genetic studies. The Java medaka, , is one of the two species of medaka perfectly adapted for living in brackish/sea-waters. Being an important component of the mangrove ecosystem, is also used as a valuable marine test-fish for ecotoxicology studies. Here, we sequenced and assembled the whole genome of , and anticipate this resource will be catalytic for a wide range of comparative genomic, phylogenetic and functional studies. Complementary sequencing approaches including long-read technology and data integration with a genetic map allowed the final assembly of 908 Mbp of the genome. Further analyses estimate that the genome contains 33% of repeat sequences and has a heterozygosity of 0.96%. The achieved draft assembly contains 525 scaffolds with a total length of 809.7 Mbp, a N50 of 6,3 Mbp and a L50 of 37 scaffolds. We identified 21454 predicted transcripts for a total transcriptome size of 57, 146, 583 bps. We provide here a high-quality chromosome scale draft genome assembly of the euryhaline Javafish medaka (321 scaffolds anchored on 24 chromosomes (representing 97.7% of the total bases)), and give emphasis on the evolutionary adaptation to salinity.
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http://dx.doi.org/10.1534/g3.119.400725DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7056978PMC
March 2020

Independent Origin of XY and ZW Sex Determination Mechanisms in Mosquitofish Sister Species.

Genetics 2020 01 8;214(1):193-209. Epub 2019 Nov 8.

Physiological Chemistry, Biocenter, University of Wuerzburg, 97074, Germany

Fish are known for the outstanding variety of their sex determination mechanisms and sex chromosome systems. The western () and eastern mosquitofish () are sister species for which different sex determination mechanisms have been described: ZZ/ZW for and XX/XY for Here, we carried out restriction-site associated DNA (RAD-) and pool sequencing (Pool-seq) to characterize the sex chromosomes of both species. We found that the ZW chromosomes of females and the XY chromosomes of males correspond to different linkage groups, and thus evolved independently from separate autosomes. In interspecific hybrids, the Y chromosome is dominant over the W chromosome, and X is dominant over Z. In , we identified a candidate region for the Y-linked melanic pigmentation locus, a rare male phenotype that constitutes a potentially sexually antagonistic trait and is associated with other such characteristics, , large body size and aggressive behavior. We developed a SNP-based marker in the Y-linked allele of (), which was linked to melanism in all tested populations. This locus represents an example for a color locus that is located in close proximity to a putative sex determiner, and most likely substantially contributed to the evolution of the Y.
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http://dx.doi.org/10.1534/genetics.119.302698DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6944411PMC
January 2020

Identification of the master sex determining gene in Northern pike (Esox lucius) reveals restricted sex chromosome differentiation.

PLoS Genet 2019 08 22;15(8):e1008013. Epub 2019 Aug 22.

INRA, UR1037 LPGP, Campus de Beaulieu, Rennes, France.

Teleost fishes, thanks to their rapid evolution of sex determination mechanisms, provide remarkable opportunities to study the formation of sex chromosomes and the mechanisms driving the birth of new master sex determining (MSD) genes. However, the evolutionary interplay between the sex chromosomes and the MSD genes they harbor is rather unexplored. We characterized a male-specific duplicate of the anti-Müllerian hormone (amh) as the MSD gene in Northern Pike (Esox lucius), using genomic and expression evidence as well as by loss-of-function and gain-of-function experiments. Using RAD-Sequencing from a family panel, we identified Linkage Group (LG) 24 as the sex chromosome and positioned the sex locus in its sub-telomeric region. Furthermore, we demonstrated that this MSD originated from an ancient duplication of the autosomal amh gene, which was subsequently translocated to LG24. Using sex-specific pooled genome sequencing and a new male genome sequence assembled using Nanopore long reads, we also characterized the differentiation of the X and Y chromosomes, revealing a small male-specific insertion containing the MSD gene and a limited region with reduced recombination. Our study reveals an unexpectedly low level of differentiation between a pair of sex chromosomes harboring an old MSD gene in a wild teleost fish population, and highlights both the pivotal role of genes from the amh pathway in sex determination, as well as the importance of gene duplication as a mechanism driving the turnover of sex chromosomes in this clade.
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http://dx.doi.org/10.1371/journal.pgen.1008013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6726246PMC
August 2019

ISoLDE: a data-driven statistical method for the inference of allelic imbalance in datasets with reciprocal crosses.

Bioinformatics 2020 01;36(2):504-513

Institut de Génomique Fonctionnelle, IGF, Univ. Montpellier, CNRS, INSERM, 34094 Montpellier, France.

Motivation: Allelic imbalance (AI), i.e. the unequal expression of the alleles of the same gene in a single cell, affects a subset of genes in diploid organisms. One prominent example of AI is parental genomic imprinting, which results in parent-of-origin-dependent, mono-allelic expression of a limited number of genes in metatherian and eutherian mammals and in angiosperms. Currently available methods for identifying AI rely on data modeling and come with the associated limitations.

Results: We have designed ISoLDE (Integrative Statistics of alleLe Dependent Expression), a novel nonparametric statistical method that takes into account both AI and the characteristics of RNA-seq data to infer allelic expression bias when at least two biological replicates are available for reciprocal crosses. ISoLDE learns the distribution of a specific test statistic from the data and calls genes 'allelically imbalanced', 'bi-allelically expressed' or 'undetermined'. Depending on the number of replicates, predefined thresholds or permutations are used to make calls. We benchmarked ISoLDE against published methods, and showed that ISoLDE compared favorably with respect to sensitivity, specificity and robustness to the number of replicates. Using ISoLDE on different RNA-seq datasets generated from hybrid mouse tissues, we did not discover novel imprinted genes (IGs), confirming the most conservative estimations of IG number.

Availability And Implementation: ISoLDE has been implemented as a Bioconductor package available at http://bioconductor.org/packages/ISoLDE/.

Supplementary Information: Supplementary data are available at Bioinformatics online.
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http://dx.doi.org/10.1093/bioinformatics/btz564DOI Listing
January 2020

What makes a bad egg? Egg transcriptome reveals dysregulation of translational machinery and novel fertility genes important for fertilization.

BMC Genomics 2019 Jul 15;20(1):584. Epub 2019 Jul 15.

INRA, Laboratoire de Physiologie et Génomique des poissons, Campus de Beaulieu, F-35042, Rennes cedex, France.

Background: Egg quality can be defined as the egg ability to be fertilized and subsequently develop into a normal embryo. Previous research has shed light on factors that can influence egg quality. Large gaps however remain including a comprehensive view of what makes a bad egg. Initial development of the embryo relies on maternally-inherited molecules, such as transcripts, deposited in the egg during its formation. Bad egg quality is therefore susceptible to be associated with alteration or dysregulation of maternally-inherited transcripts. We performed transcriptome analysis on a large number (N = 136) of zebrafish egg clutches, each clutch being split to monitor developmental success and perform transcriptome analysis in parallel. We aimed at drawing a molecular portrait of the egg in order to characterize the relation between egg transcriptome and developmental success and to subsequently identify new candidate genes involved in fertility.

Results: We identified 66 transcript that were differentially abundant in eggs of contrasted phenotype (low or high developmental success). Statistical modeling using partial least squares regression and genetics algorithm demonstrated that gene signatures from transcriptomic data can be used to predict developmental success. The identity and function of differentially expressed genes indicate a major dysregulation of genes of the translational machinery in poor quality eggs. Two genes, otulina and slc29a1a, predominantly expressed in the ovary and dysregulated in poor quality eggs were further investigated using CRISPR/Cas9 mediated genome editing. Mutants of each gene revealed remarkable subfertility whereby the majority of their eggs were unfertilizable. The Wnt pathway appeared to be dysregulated in the otulina mutant-derived eggs.

Conclusions: Here we show that egg transcriptome contains molecular signatures, which can be used to predict developmental success. Our results also indicate that poor egg quality in zebrafish is associated with a dysregulation of (i) the translational machinery genes and (ii) novel fertility genes, otulina and slc29a1a, playing an important role for fertilization. Together, our observations highlight the diversity of the possible causes of egg quality defects and reveal mechanisms of maternal origin behind the lack of fertilization and early embryonic failures that can occur under normal reproduction conditions.
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http://dx.doi.org/10.1186/s12864-019-5930-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6631549PMC
July 2019

The genome of the arapaima (Arapaima gigas) provides insights into gigantism, fast growth and chromosomal sex determination system.

Sci Rep 2019 03 28;9(1):5293. Epub 2019 Mar 28.

University of Wuerzburg, Physiological Chemistry, Biocenter, 97074, Wuerzburg, Germany.

We have sequenced the genome of the largest freshwater fish species of the world, the arapaima. Analysis of gene family dynamics and signatures of positive selection identified genes involved in the specific adaptations and unique features of this iconic species, in particular it's large size and fast growth. Genome sequences from both sexes combined with RAD-tag analyses from other males and females led to the isolation of male-specific scaffolds and supports an XY sex determination system in arapaima. Whole transcriptome sequencing showed that the product of the gland-like secretory organ on the head surface of males and females may not only provide nutritional fluid for sex-unbiased parental care, but that the organ itself has a more specific function in males, which engage more in parental care.
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http://dx.doi.org/10.1038/s41598-019-41457-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6439221PMC
March 2019

Cerebral Cortex Generated from Pluripotent Stem Cells to Model Corticogenesis and Rebuild Cortical Circuits: In Vitro Veritas?

Stem Cells Dev 2019 03 20;28(6):361-369. Epub 2019 Feb 20.

Institut de Génomique Fonctionnelle, IGF, CNRS, INSERM, Université de Montpellier, Montpellier, France.

Organoids and cells generated in vitro from pluripotent stem cells (PSCs) are considered to be robust models of development and a conceivable source of transplants for putative cell therapy. However, a fundamental question about organoids and cells generated from PSCs is as follows: do they faithfully reproduce the in vivo tissue they are supposed to mimic and replace? This question is particularly relevant to complex tissues such as the cerebral cortex. In this review, we have tackled this issue by comparing cerebral cortices generated in vitro from PSCs to the in vivo cortex, with a particular focus on their respective cellular composition, molecular and epigenetic signatures, and brain connectivity. In short, in vitro cortex generated from PSCs reproduces most of the cardinal features of the in vivo cortex, including temporal corticogenesis and connectivity when PSC-derived cortical cells are grafted in recipient mouse cortex. However, compared to in vivo cortex, in vitro cortex lacks microglia and blood vessels and is less mature. Recent experiments show that the brain of the transplanted host provides these missing cell types together with an environment that promotes the synaptic maturation of the cortical transplant. Taken together, these data suggest that corticogenesis is largely intrinsic and well recapitulated in vitro, while the full maturation of cortical cells requires additional environmental clues. Finally, we propose some lines of work to improve corticogenesis from PSCs as a tool to model corticogenesis and rebuild cortical circuits.
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http://dx.doi.org/10.1089/scd.2018.0233DOI Listing
March 2019

High-salt-recovered sequences are associated with the active chromosomal compartment and with large ribonucleoprotein complexes including nuclear bodies.

Genome Res 2018 11 4;28(11):1733-1746. Epub 2018 Oct 4.

IGMM, Université de Montpellier, CNRS, F-34293, Montpellier, France.

The mammalian cell nucleus contains numerous discrete suborganelles named nuclear bodies. While recruitment of specific genomic regions into these large ribonucleoprotein (RNP) complexes critically contributes to higher-order functional chromatin organization, such regions remain ill-defined. We have developed the high-salt-recovered sequences-sequencing (HRS-seq) method, a straightforward genome-wide approach whereby we isolated and sequenced genomic regions associated with large high-salt insoluble RNP complexes. By using mouse embryonic stem cells (ESCs), we showed that these regions essentially correspond to the most highly expressed genes, and to -regulatory sequences like super-enhancers, that belong to the active A chromosomal compartment. They include both cell-type-specific genes, such as pluripotency genes in ESCs, and housekeeping genes associated with nuclear bodies, such as histone and snRNA genes that are central components of Histone Locus Bodies and Cajal bodies. We conclude that HRSs are associated with the active chromosomal compartment and with large RNP complexes including nuclear bodies. Association of such chromosomal regions with nuclear bodies is in agreement with the recently proposed phase separation model for transcription control and might thus play a central role in organizing the active chromosomal compartment in mammals.
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http://dx.doi.org/10.1101/gr.237073.118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6211644PMC
November 2018

Mouse Parthenogenetic Embryonic Stem Cells with Biparental-Like Expression of Imprinted Genes Generate Cortical-Like Neurons That Integrate into the Injured Adult Cerebral Cortex.

Stem Cells 2018 02 10;36(2):192-205. Epub 2017 Nov 10.

Institut de Génomique Fonctionnelle, IGF, CNRS, INSERM, Univ. Montpellier, Montpellier, France.

One strategy for stem cell-based therapy of the cerebral cortex involves the generation and transplantation of functional, histocompatible cortical-like neurons from embryonic stem cells (ESCs). Diploid parthenogenetic Pg-ESCs have recently emerged as a promising source of histocompatible ESC derivatives for organ regeneration but their utility for cerebral cortex therapy is unknown. A major concern with Pg-ESCs is genomic imprinting. In contrast with biparental Bp-ESCs derived from fertilized oocytes, Pg-ESCs harbor two maternal genomes but no sperm-derived genome. Pg-ESCs are therefore expected to have aberrant expression levels of maternally expressed (MEGs) and paternally expressed (PEGs) imprinted genes. Given the roles of imprinted genes in brain development, tissue homeostasis and cancer, their deregulation in Pg-ESCs might be incompatible with therapy. Here, we report that, unexpectedly, only one gene out of 7 MEGs and 12 PEGs was differentially expressed between Pg-ESCs and Bp-ESCs while 13 were differentially expressed between androgenetic Ag-ESCs and Bp-ESCs, indicating that Pg-ESCs but not Ag-ESCs, have a Bp-like imprinting compatible with therapy. In vitro, Pg-ESCs generated cortical-like progenitors and electrophysiologically active glutamatergic neurons that maintained the Bp-like expression levels for most imprinted genes. In vivo, Pg-ESCs participated to the cortical lineage in fetal chimeras. Finally, transplanted Pg-ESC derivatives integrated into the injured adult cortex and sent axonal projections in the host brain. In conclusion, mouse Pg-ESCs generate functional cortical-like neurons with Bp-like imprinting and their derivatives properly integrate into both the embryonic cortex and the injured adult cortex. Collectively, our data support the utility of Pg-ESCs for cortical therapy. Stem Cells 2018;36:192-205.
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http://dx.doi.org/10.1002/stem.2721DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5785436PMC
February 2018

Identification of Plagl1/Zac1 binding sites and target genes establishes its role in the regulation of extracellular matrix genes and the imprinted gene network.

Nucleic Acids Res 2017 Oct;45(18):10466-10480

Institut de Génomique Fonctionnelle, IGF, CNRS, INSERM, Univ. Montpellier, F-34094 Montpellier, France.

PLAGL1/ZAC1 undergoes parental genomic imprinting, is paternally expressed, and is a member of the imprinted gene network (IGN). It encodes a zinc finger transcription factor with anti-proliferative activity and is a candidate tumor suppressor gene on 6q24 whose expression is frequently lost in various neoplasms. Conversely, gain of PLAGL1 function is responsible for transient neonatal diabetes mellitus, a rare genetic disease that results from defective pancreas development. In the present work, we showed that Plagl1 up-regulation was not associated with DNA damage-induced cell cycle arrest. It was rather associated with physiological cell cycle exit that occurred with contact inhibition, growth factor withdrawal, or cell differentiation. To gain insights into Plagl1 mechanism of action, we identified Plagl1 target genes by combining chromatin immunoprecipitation and genome-wide transcriptomics in transfected cell lines. Plagl1-elicited gene regulation correlated with multiple binding to the proximal promoter region through a GC-rich motif. Plagl1 target genes included numerous genes involved in signaling, cell adhesion, and extracellular matrix composition, including collagens. Plagl1 targets also included 22% of the 409 genes that make up the IGN. Altogether, this work identified Plagl1 as a transcription factor that coordinated the regulation of a subset of IGN genes and controlled extracellular matrix composition.
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http://dx.doi.org/10.1093/nar/gkx672DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5737700PMC
October 2017

Evolution of gene expression after whole-genome duplication: New insights from the spotted gar genome.

J Exp Zool B Mol Dev Evol 2017 Nov 25;328(7):709-721. Epub 2017 Sep 25.

INRA, UR1037 LPGP, Campus de Beaulieu, Rennes, France.

Whole-genome duplications (WGDs) are important evolutionary events. Our understanding of underlying mechanisms, including the evolution of duplicated genes after WGD, however, remains incomplete. Teleost fish experienced a common WGD (teleost-specific genome duplication, or TGD) followed by a dramatic adaptive radiation leading to more than half of all vertebrate species. The analysis of gene expression patterns following TGD at the genome level has been limited by the lack of suitable genomic resources. The recent concomitant release of the genome sequence of spotted gar (a representative of holosteans, the closest-related lineage of teleosts that lacks the TGD) and the tissue-specific gene expression repertoires of over 20 holostean and teleostean fish species, including spotted gar, zebrafish, and medaka (the PhyloFish project), offers a unique opportunity to study the evolution of gene expression following TGD in teleosts. We show that most TGD duplicates gained their current status (loss of one duplicate gene or retention of both duplicates) relatively rapidly after TGD (i.e., prior to the divergence of medaka and zebrafish lineages). The loss of one duplicate is the most common fate after TGD with a probability of approximately 80%. In addition, the fate of duplicate genes after TGD, including subfunctionalization, neofunctionalization, or retention of two "similar" copies occurred not only before but also after the divergence of species tested, in consistency with a role of the TGD in speciation and/or evolution of gene function. Finally, we report novel cases of TGD ohnolog subfunctionalization and neofunctionalization that further illustrate the importance of these processes.
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http://dx.doi.org/10.1002/jez.b.22770DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5679426PMC
November 2017

Gene evolution and gene expression after whole genome duplication in fish: the PhyloFish database.

BMC Genomics 2016 05 18;17:368. Epub 2016 May 18.

INRA, Laboratoire de Physiologie et Génomique des poissons, Campus de Beaulieu, F-35042, Rennes cedex, France.

With more than 30,000 species, ray-finned fish represent approximately half of vertebrates. The evolution of ray-finned fish was impacted by several whole genome duplication (WGD) events including a teleost-specific WGD event (TGD) that occurred at the root of the teleost lineage about 350 million years ago (Mya) and more recent WGD events in salmonids, carps, suckers and others. In plants and animals, WGD events are associated with adaptive radiations and evolutionary innovations. WGD-spurred innovation may be especially relevant in the case of teleost fish, which colonized a wide diversity of habitats on earth, including many extreme environments. Fish biodiversity, the use of fish models for human medicine and ecological studies, and the importance of fish in human nutrition, fuel an important need for the characterization of gene expression repertoires and corresponding evolutionary histories of ray-finned fish genes. To this aim, we performed transcriptome analyses and developed the PhyloFish database to provide (i) de novo assembled gene repertoires in 23 different ray-finned fish species including two holosteans (i.e. a group that diverged from teleosts before TGD) and 21 teleosts (including six salmonids), and (ii) gene expression levels in ten different tissues and organs (and embryos for many) in the same species. This resource was generated using a common deep RNA sequencing protocol to obtain the most exhaustive gene repertoire possible in each species that allows between-species comparisons to study the evolution of gene expression in different lineages. The PhyloFish database described here can be accessed and searched using RNAbrowse, a simple and efficient solution to give access to RNA-seq de novo assembled transcripts.
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http://dx.doi.org/10.1186/s12864-016-2709-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4870732PMC
May 2016

In Vitro Corticogenesis from Embryonic Stem Cells Recapitulates the In Vivo Epigenetic Control of Imprinted Gene Expression.

Cereb Cortex 2017 03;27(3):2418-2433

Institut de Génomique Fonctionnelle (IGF), CNRS UMR5203, INSERM U1191, Université de Montpellier, Montpellier, France.

In vitro corticogenesis from embryonic stem cells (ESCs) is an attractive model of cortical development and a promising tool for cortical therapy. It is unknown to which extent epigenetic mechanisms crucial for cortex development and function, such as parental genomic imprinting, are recapitulated by in vitro corticogenesis. Here, using genome-wide transcriptomic and methylation analyses on hybrid mouse tissues and cells, we find a high concordance of imprinting status between in vivo and ESC-derived cortices. Notably, in vitro corticogenesis strictly reproduced the in vivo parent-of-origin-dependent expression of 41 imprinted genes (IGs), including Mest and Cdkn1c known to control corticogenesis. Parent-of-origin-dependent DNA methylation was also conserved at 14 of 18 imprinted differentially methylated regions. The least concordant imprinted locus was Gpr1-Zdbf2, where the aberrant bi-allelic expression of Zdbf2 and Adam23 was concomitant with a gain of methylation on the maternal allele in vitro. Combined, our data argue for a broad conservation of the epigenetic mechanisms at imprinted loci in cortical cells derived from ESCs. We propose that in vitro corticogenesis helps to define the still poorly understood mechanisms that regulate imprinting in the brain and the roles of IGs in cortical development.
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http://dx.doi.org/10.1093/cercor/bhw102DOI Listing
March 2017

Zac1 Regulates the Differentiation and Migration of Neocortical Neurons via Pac1.

J Neurosci 2015 Sep;35(39):13430-47

Departments of Biochemistry and Molecular Biology, Alberta Children's Hospital Research Institute, Hotchkiss Brain Institute, University of Calgary, Calgary, Alberta, Canada, T2N 4N1,

Imprinted genes are dosage sensitive, and their dysregulated expression is linked to disorders of growth and proliferation, including fetal and postnatal growth restriction. Common sequelae of growth disorders include neurodevelopmental defects, some of which are indirectly related to placental insufficiency. However, several growth-associated imprinted genes are also expressed in the embryonic CNS, in which their aberrant expression may more directly affect neurodevelopment. To test whether growth-associated genes influence neural lineage progression, we focused on the maternally imprinted gene Zac1. In humans, either loss or gain of ZAC1 expression is associated with reduced growth rates and intellectual disability. To test whether increased Zac1 expression directly perturbs neurodevelopment, we misexpressed Zac1 in murine neocortical progenitors. The effects were striking: Zac1 delayed the transition of apical radial glial cells to basal intermediate neuronal progenitors and postponed their subsequent differentiation into neurons. Zac1 misexpression also blocked neuronal migration, with Zac1-overexpressing neurons pausing more frequently and forming fewer neurite branches during the period when locomoting neurons undergo dynamic morphological transitions. Similar, albeit less striking, neuronal migration and morphological defects were observed on Zac1 knockdown, indicating that Zac1 levels must be regulated precisely. Finally, Zac1 controlled neuronal migration by regulating Pac1 transcription, a receptor for the neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP). Pac1 and Zac1 loss- and gain-of-function presented as phenocopies, and overexpression of Pac1 rescued the Zac1 knockdown neuronal migration phenotype. Thus, dysregulated Zac1 expression has striking consequences on neocortical development, suggesting that misexpression of this transcription factor in the brain in certain growth disorders may contribute to neurocognitive deficits. Significance statement: Altered expression of imprinted genes is linked to cognitive dysfunction and neuropsychological disorders, such as Angelman and Prader-Willi syndromes, and autism spectrum disorder. Mouse models have also revealed the importance of imprinting for brain development, with chimeras generated with parthenogenetic (two maternal chromosomes) or androgenetic (two paternal chromosomes) cells displaying altered brain sizes and cellular defects. Despite these striking phenotypes, only a handful of imprinted genes are known or suspected to regulate brain development (e.g., Dlk1, Peg3, Ube3a, necdin, and Grb10). Herein we show that the maternally imprinted gene Zac1 is a critical regulator of neocortical development. Our studies are relevant because loss of 6q24 maternal imprinting in humans results in elevated ZAC1 expression, which has been associated with neurocognitive defects.
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http://dx.doi.org/10.1523/JNEUROSCI.0777-15.2015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6605482PMC
September 2015

A systems-level approach to parental genomic imprinting: the imprinted gene network includes extracellular matrix genes and regulates cell cycle exit and differentiation.

Genome Res 2015 Mar 22;25(3):353-67. Epub 2015 Jan 22.

Institut de Genomique Fonctionnelle, Montpellier 34094, France; CNRS, UMR 5203, Montpellier 34094, France; INSERM, U661, Montpellier 34094, France; Faculté des Sciences, Université de Montpellier, Montpellier 34095, France; MGX-Montpellier GenomiX, Montpellier 34094, France

Genomic imprinting is an epigenetic mechanism that restrains the expression of ∼ 100 eutherian genes in a parent-of-origin-specific manner. The reason for this selective targeting of genes with seemingly disparate molecular functions is unclear. In the present work, we show that imprinted genes are coexpressed in a network that is regulated at the transition from proliferation to quiescence and differentiation during fibroblast cell cycle withdrawal, adipogenesis in vitro, and muscle regeneration in vivo. Imprinted gene regulation is not linked to alteration of DNA methylation or to perturbation of monoallelic, parent-of-origin-dependent expression. Overexpression and knockdown of imprinted gene expression alters the sensitivity of preadipocytes to contact inhibition and adipogenic differentiation. In silico and in cellulo experiments showed that the imprinted gene network includes biallelically expressed, nonimprinted genes. These control the extracellular matrix composition, cell adhesion, cell junction, and extracellular matrix-activated and growth factor-activated signaling. These observations show that imprinted genes share a common biological process that may account for their seemingly diverse roles in embryonic development, obesity, diabetes, muscle physiology, and neoplasm.
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http://dx.doi.org/10.1101/gr.175919.114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4352888PMC
March 2015

ICR noncoding RNA expression controls imprinting and DNA replication at the Dlk1-Dio3 domain.

Dev Cell 2014 Oct 25;31(1):19-33. Epub 2014 Sep 25.

Institute of Molecular Genetics (IGMM), CNRS UMR5535 and University of Montpellier, 1919 Route de Mende, Montpellier 34293, France. Electronic address:

Imprinted genes play essential roles in development, and their allelic expression is mediated by imprinting control regions (ICRs). The Dlk1-Dio3 locus is among the few imprinted domains controlled by a paternally methylated ICR. The unmethylated maternal copy activates imprinted expression early in development through an unknown mechanism. We find that in mouse embryonic stem cells (ESCs) and in blastocysts, this function is linked to maternal, bidirectional expression of noncoding RNAs (ncRNAs) from the ICR. Disruption of ICR ncRNA expression in ESCs affected gene expression in cis, led to acquisition of aberrant histone and DNA methylation, delayed replication timing along the domain on the maternal chromosome, and changed its subnuclear localization. The epigenetic alterations persisted during differentiation and affected the neurogenic potential of the stem cells. Our data indicate that monoallelic expression at an ICR of enhancer RNA-like ncRNAs controls imprinted gene expression, epigenetic maintenance processes, and DNA replication in embryonic cells.
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http://dx.doi.org/10.1016/j.devcel.2014.08.009DOI Listing
October 2014

Systems medicine approaches for the definition of complex phenotypes in chronic diseases and ageing. From concept to implementation and policies.

Curr Pharm Des 2014 ;20(38):5928-44

Chronic diseases are diseases of long duration and slow progression. Major NCDs (cardiovascular diseases, cancer, chronic respiratory diseases, diabetes, rheumatologic diseases and mental health) represent the predominant health problem of the Century. The prevention and control of NCDs are the priority of the World Health Organization 2008 Action Plan, the United Nations 2010 Resolution and the European Union 2010 Council. The novel trend for the management of NCDs is evolving towards integrative, holistic approaches. NCDs are intertwined with ageing. The European Innovation Partnership on Active and Healthy Ageing (EIP on AHA) has prioritised NCDs. To tackle them in their totality in order to reduce their burden and societal impact, it is proposed that NCDs should be considered as a single expression of disease with different risk factors and entities. An innovative integrated health system built around systems medicine and strategic partnerships is proposed to combat NCDs. It includes (i) understanding the social, economic, environmental, genetic determinants, as well as the molecular and cellular mechanisms underlying NCDs; (ii) primary care and practice-based interprofessional collaboration; (iii) carefully phenotyped patients; (iv) development of unbiased and accurate biomarkers for comorbidities, severity and follow up of patients; (v) socio-economic science; (vi) development of guidelines; (vii) training; and (viii) policy decisions. The results could be applicable to all countries and adapted to local needs, economy and health systems. This paper reviews the complexity of NCDs intertwined with ageing. It gives an overview of the problem and proposes two practical examples of systems medicine (MeDALL) applied to allergy and to NCD co-morbidities (MACVIA-LR, Reference Site of the European Innovation Partnership on Active and Healthy Ageing).
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http://dx.doi.org/10.2174/1381612820666140314115505DOI Listing
June 2015

Expression at the imprinted dlk1-gtl2 locus is regulated by proneural genes in the developing telencephalon.

PLoS One 2012 6;7(11):e48675. Epub 2012 Nov 6.

IRIBHM (Institute for Interdisciplinary Research), Université Libre de Bruxelles (ULB), Brussels, Belgium.

Imprinting is an epigenetic mechanism that restrains the expression of about 100 genes to one allele depending on its parental origin. Several imprinted genes are implicated in neurodevelopmental brain disorders, such as autism, Angelman, and Prader-Willi syndromes. However, how expression of these imprinted genes is regulated during neural development is poorly understood. Here, using single and double KO animals for the transcription factors Neurogenin2 (Ngn2) and Achaete-scute homolog 1 (Ascl1), we found that the expression of a specific subset of imprinted genes is controlled by these proneural genes. Using in situ hybridization and quantitative PCR, we determined that five imprinted transcripts situated at the Dlk1-Gtl2 locus (Dlk1, Gtl2, Mirg, Rian, Rtl1) are upregulated in the dorsal telencephalon of Ngn2 KO mice. This suggests that Ngn2 influences the expression of the entire Dlk1-Gtl2 locus, independently of the parental origin of the transcripts. Interestingly 14 other imprinted genes situated at other imprinted loci were not affected by the loss of Ngn2. Finally, using Ngn2/Ascl1 double KO mice, we show that the upregulation of genes at the Dlk1-Gtl2 locus in Ngn2 KO animals requires a functional copy of Ascl1. Our data suggest a complex interplay between proneural genes in the developing forebrain that control the level of expression at the imprinted Dlk1-Gtl2 locus (but not of other imprinted genes). This raises the possibility that the transcripts of this selective locus participate in the biological effects of proneural genes in the developing telencephalon.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0048675PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3490856PMC
April 2013

Double Myod and Igf2 inactivation promotes brown adipose tissue development by increasing Prdm16 expression.

FASEB J 2012 Nov 2;26(11):4584-91. Epub 2012 Aug 2.

Genetics and Development Department, Institut National de Santé et de Recherche Médicale (INSERM) U567, Centre National de Recherche Scientifique (CNRS) Unité Mixte de Recherche 8104, University of Paris Descartes, Institut Cochin, Paris, France.

Brown fat or brown adipose tissue (BAT), found in newborn mammals as small depots localized in the interscapular region, plays a prominent role in regulating thermogenesis perinatally. The physiological importance of functional BAT has been recently reasserted in human adults. Because myoblasts and adipoblasts emerge from a common mesodermal precursor, we investigated developmental determination and the reciprocal relationship between muscle and adipocyte commitment. Here we show that a mutant mouse defective for both Igf2 and Myod genes exhibits massive BAT hypertrophy compared with wild-type and single-mutant newborns. The increased adipocyte proliferation in BAT of double-mutant newborns was associated with overexpression of the brown fat-specific marker Ucp1. More strikingly, expression of the master key gene Prdm16 involved in the switch between myogenic and brown adipogenic lineages was drastically enhanced. We further demonstrate that concomitant Myod and Igf2 inactivation accelerates differentiation of a brown preadipocyte cell line and induces lipid accumulation and increased Ucp1 and Prdm16 expression. This in vitro approach brings additional support for the implication of both Myod and Igf2 in BAT development. These results provide the first in vivo evidence that a myogenic regulator together with a growth factor act simultaneously but through independent pathways to repress Prdm16, which opens potential therapeutic perspectives for human metabolic disorders.
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http://dx.doi.org/10.1096/fj.12-208496DOI Listing
November 2012

Essential requirement for β-arrestin2 in mouse intestinal tumors with elevated Wnt signaling.

Proc Natl Acad Sci U S A 2012 Feb 6;109(8):3047-52. Epub 2012 Feb 6.

Centre National de la Recherche Scientifique Unité Mixte de Recherche 5203, Institut de Génomique Fonctionnelle, F-34000 Montpellier, France.

β-Arrestins (Arrb) participate in the regulation of multiple signaling pathways, including Wnt/β-catenin, the major actor in human colorectal cancer initiation. To better understand the roles of Arrb in intestinal tumorigenesis, a reverse genetic approach (Arrb(-/-)) and in vivo siRNA treatment were used in Apc(Δ14/+) mice. Mice with Arrb2 depletion (knockout and siRNA) developed only 33% of the tumors detected in their Arrb2-WT littermates, whereas Arrb1 depletion remained without significant effect. These remaining tumors grow normally and are essentially Arrb2-independent. Unsupervised hierarchical clustering analysis showed that they clustered with 25% of Apc(Δ14/+);Arrb2(+/+) tumors. Genes overexpressed in this subset reflect a high interaction with the immune system, whereas those overexpressed in Arrb2-dependent tumors are predominantly involved in Wnt signaling, cell adhesion, migration, and extracellular matrix remodeling. The involvement of Arrb2 in intestinal tumor development via the regulation of the Wnt pathway is supported by ex vivo and in vitro experiments using either tumors from Apc(Δ14/+) mice or murine Apc(Min/+) cells. Indeed, Arrb2 siRNAs decreased the expression of Wnt target genes in cells isolated from 12 of 18 tumors from Apc(Δ14/+) mice. In Apc(Min/+) cells, Arrb2 siRNAs completely reversed the increased Wnt activity and colony formation in soft agar induced by Apc siRNA treatment, whereas they did not affect these parameters in basal conditions or in cells expressing constitutively active β-catenin. We demonstrate that Arrb2 is essential for the initiation and growth of intestinal tumors displaying elevated Wnt pathway activity and identify a previously unsuspected molecular heterogeneity among tumors induced by truncating Apc mutations.
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http://dx.doi.org/10.1073/pnas.1109457109DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3286958PMC
February 2012

Genome-wide profiling of G protein-coupled receptors in cerebellar granule neurons using high-throughput, real-time PCR.

BMC Genomics 2011 May 16;12:241. Epub 2011 May 16.

Institut de Genomique Fonctionnelle, 141 rue de la cardonille, F-34094 Montpellier Cedex 05, France.

Background: G protein-coupled receptors (GPCRs) are major players in cell communication, regulate a whole range of physiological functions during development and throughout adult life, are affected in numerous pathological situations, and constitute so far the largest class of drugable targets for human diseases. The corresponding genes are usually expressed at low levels, making accurate, genome-wide quantification of their expression levels a challenging task using microarrays.

Results: We first draw an inventory of all endo-GPCRs encoded in the murine genome. To profile GPCRs genome-wide accurately, sensitively, comprehensively, and cost-effectively, we designed and validated a collection of primers that we used in quantitative RT-PCR experiments. We experimentally validated a statistical approach to analyze genome-wide, real-time PCR data. To illustrate the usefulness of this approach, we determined the repertoire of GPCRs expressed in cerebellar granule neurons and neuroblasts during postnatal development.

Conclusions: We identified tens of GPCRs that were not detected previously in this cell type; these GPCRs represent novel candidate players in the development and survival of cerebellar granule neurons. The sequences of primers used in this study are freely available to those interested in quantifying GPCR expression comprehensively.
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http://dx.doi.org/10.1186/1471-2164-12-241DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3111393PMC
May 2011

The 'PUCE CAFE' Project: the first 15K coffee microarray, a new tool for discovering candidate genes correlated to agronomic and quality traits.

BMC Genomics 2011 Jan 5;12. Epub 2011 Jan 5.

Nestlé R&D Tours, 101 Avenue Gustave Eiffel, Notre Dame d'Oé, BP 49716, 37097 Tours Cedex 2, France.

Background: Understanding the genetic elements that contribute to key aspects of coffee biology will have an impact on future agronomical improvements for this economically important tree. During the past years, EST collections were generated in Coffee, opening the possibility to create new tools for functional genomics.

Results: The "PUCE CAFE" Project, organized by the scientific consortium NESTLE/IRD/CIRAD, has developed an oligo-based microarray using 15,721 unigenes derived from published coffee EST sequences mostly obtained from different stages of fruit development and leaves in Coffea Canephora (Robusta). Hybridizations for two independent experiments served to compare global gene expression profiles in three types of tissue matter (mature beans, leaves and flowers) in C. canephora as well as in the leaves of three different coffee species (C. canephora, C. eugenoides and C. arabica). Microarray construction, statistical analyses and validation by Q-PCR analysis are presented in this study.

Conclusion: We have generated the first 15 K coffee array during this PUCE CAFE project, granted by Génoplante (the French consortium for plant genomics). This new tool will help study functional genomics in a wide range of experiments on various plant tissues, such as analyzing bean maturation or resistance to pathogens or drought. Furthermore, the use of this array has proven to be valid in different coffee species (diploid or tetraploid), drastically enlarging its impact for high-throughput gene expression in the community of coffee research.
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http://dx.doi.org/10.1186/1471-2164-12-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3025959PMC
January 2011

Viral induction of Zac1b through TLR3- and IRF3-dependent pathways.

Mol Immunol 2010 Nov-Dec;48(1-3):119-27. Epub 2010 Oct 13.

Laboratory of Cellular and Molecular Physiology, GIGA-Research, University of Liege, Liege, Belgium.

Zinc finger protein regulator of apoptosis and cell cycle arrest (Zac1) is a transcription factor able to induce apoptosis or cell cycle arrest through independent pathways. In spite of the important potential functions attributed to Zac1, little is known of its physiological regulation and biological function. We discovered that variant Zac1b was expressed in murine embryonic fibroblasts (MEFs) treated with polyriboinosinic polyribocytidylic acid [poly(I:C)], a synthetic double-stranded RNA. This regulation occurred mainly through Toll-Like Receptor 3 (TLR3)- and Interferon Regulatory Factor 3 (IRF3)-dependent pathways. As TLR3 and IRF3 are central activators of antiviral immunity, we hypothesized that Zac1 may be implicated in antiviral responses. In line with this notion, we observed that Zac1b was expressed in MEFs infected with Encephalomyocarditis virus (EMCV). We also observed that Zac1-deficient MEFs were less sensitive to EMCV-induced cell death than wild-type MEFs. However, Zac1 gene inactivation had no effect on the survival of mice infected with EMCV. In conclusion, this study describes for the first time a transcriptional regulation of Zac1b, induced by synthetic dsRNA and RNA viruses, the functional significance of which remains to be further investigated.
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http://dx.doi.org/10.1016/j.molimm.2010.09.004DOI Listing
January 2011

In vitro fertilization and embryo culture strongly impact the placental transcriptome in the mouse model.

PLoS One 2010 Feb 15;5(2):e9218. Epub 2010 Feb 15.

Service d'Histologie-Embryologie, Biologie de la Reproduction, Hôpital Cochin, Paris, France.

Background: Assisted Reproductive Technologies (ART) are increasingly used in humans; however, their impact is now questioned. At blastocyst stage, the trophectoderm is directly in contact with an artificial medium environment, which can impact placental development. This study was designed to carry out an in-depth analysis of the placental transcriptome after ART in mice.

Methodology/principal Findings: Blastocysts were transferred either (1) after in vivo fertilization and development (control group) or (2) after in vitro fertilization and embryo culture. Placentas were then analyzed at E10.5. Six percent of transcripts were altered at the two-fold threshold in placentas of manipulated embryos, 2/3 of transcripts being down-regulated. Strikingly, the X-chromosome harbors 11% of altered genes, 2/3 being induced. Imprinted genes were modified similarly to the X. Promoter composition analysis indicates that FOXA transcription factors may be involved in the transcriptional deregulations.

Conclusions: For the first time, our study shows that in vitro fertilization associated with embryo culture strongly modify the placental expression profile, long after embryo manipulations, meaning that the stress of artificial environment is memorized after implantation. Expression of X and imprinted genes is also greatly modulated probably to adapt to adverse conditions. Our results highlight the importance of studying human placentas from ART.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0009218PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2821408PMC
February 2010

Modulation of imprinted gene network in placenta results in normal development of in vitro manipulated mouse embryos.

Hum Mol Genet 2010 May 11;19(9):1779-90. Epub 2010 Feb 11.

Biology of Reproduction, Hospital Cochin, Paris, France

Genomic imprinting regulates the expression of a group of genes monoallelically expressed in a parent-of-origin specific manner. Allele-specific DNA methylation occurs at differentially methylated regions (DMRs) of these genes. We have previously shown that in vitro fertilization and embryo culture result in methylation defects at the imprinted H19-Igf2 locus at the blastocyst stage. The current study was designed to evaluate the consequences of these manipulations on genomic imprinting after implantation in the mouse. Blastocysts were produced following three experimental conditions: (i) embryos maintained in culture medium after in vivo fertilization or (ii) in vitro fertilization and (iii) a control group with embryos obtained after in vivo fertilization and timed mating. Blastocysts were all transplanted into pseudopregnant females. Embryos and placentas were collected on day 10.5 of development. DNA methylation patterns of the H19, Igf2, Igf2r and Dlk1-Dio3 DMRs were analyzed by quantitative pyrosequencing. In contrast to blastocyst stage, methylation profiles were normal both in embryonic and placental tissues after in vitro fertilization and culture. Expression of a selected set of imprinting genes from the recently described imprinted gene network (IGN) (including Igf2 and H19) was analyzed in placental tissues by quantitative RT-PCR. Placentas obtained after in vitro fertilization and embryo culture displayed significantly disturbed levels of H19 and Igf2 mRNA, as well as of most other genes from the IGN. As embryos were phenotypically normal, we hypothesize that the modulation of a coordinated network of imprinted genes results in a compensatory process capable of correcting potential dysfunction of placenta.
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http://dx.doi.org/10.1093/hmg/ddq059DOI Listing
May 2010

H19 acts as a trans regulator of the imprinted gene network controlling growth in mice.

Development 2009 Oct 17;136(20):3413-21. Epub 2009 Sep 17.

Genetics and Development Department, Inserm U567, CNRS UMR 8104, University of Paris Descartes, Institut Cochin, Paris, France.

The imprinted H19 gene produces a non-coding RNA of unknown function. Mice lacking H19 show an overgrowth phenotype, due to a cis effect of the H19 locus on the adjacent Igf2 gene. To explore the function of the RNA itself, we produced transgenic mice overexpressing H19. We observed postnatal growth reduction in two independent transgenic lines and detected a decrease of Igf2 expression in embryos. An extensive analysis of several other genes from the newly described imprinted gene network (IGN) was performed in both loss- and gain-of-function animals. We found that H19 deletion leads to the upregulation of several genes of the IGN. This overexpression is restored to the wild-type level by transgenic expression of H19. We therefore propose that the H19 gene participates as a trans regulator in the fine-tuning of this IGN in the mouse embryo. This is the first in vivo evidence of a functional role for the H19 RNA. Our results also bring further experimental evidence for the existence of the IGN and open new perspectives in the comprehension of the role of genomic imprinting in embryonic growth and in human imprinting pathologies.
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http://dx.doi.org/10.1242/dev.036061DOI Listing
October 2009