Barbara Pernaute

Competitive Interactions Eliminate Unfit Embryonic Stem Cells at the Onset of Differentiation

Summary A fundamental question in developmental biology is whether there are mechanisms to detect stem cells with mutations that, although not adversely affecting viability, would compromise their ability to contribute to further development. Here, we show that cell competition is a mechanism regulating the fitness of embryonic stem cells (ESCs). We find that ESCs displaying defective bone morphogenetic protein signaling or defective autophagy or that are tetraploid are eliminated at the onset of differentiation by wild-type cells. This elimination occurs in an apoptosis-dependent manner and is mediated by secreted factors. Furthermore, during this process, we find that establishment of differential c-Myc levels is critical and that c-Myc overexpression is sufficient to induce competitive behavior in ESCs. Cell competition is, therefore, a process that allows recognition and elimination of defective cells during the early stages of development and is likely to play important roles in tissue homeostasis and stem cell maintenance.

HOIP Deficiency Causes Embryonic Lethality by Aberrant TNFR1-Mediated Endothelial Cell Death

Linear ubiquitination is crucial for innate and adaptive immunity. The linear ubiquitin chain assembly complex (LUBAC), consisting of HOIL-1, HOIP, and SHARPIN, is the only known ubiquitin ligase that generates linear ubiquitin linkages. HOIP is the catalytically active LUBAC component. Here, we show that both constitutive and Tie2-Cre-driven HOIP deletion lead to aberrant endothelial cell death, resulting in defective vascularization and embryonic lethality at midgestation. Ablation of tumor necrosis factor receptor 1 (TNFR1) prevents cell death, vascularization defects, and death at midgestation. HOIP-deficient cells are more sensitive to death induction by both tumor necrosis factor (TNF) and lymphotoxin-α (LT-α), and aberrant complex-II formation is responsible for sensitization to TNFR1-mediated cell death in the absence of HOIP. Finally, we show that HOIPs catalytic activity is necessary for preventing TNF-induced cell death. Hence, LUBAC and its linear-ubiquitin-forming activity are required for maintaining vascular integrity during embryogenesis by preventing TNFR1-mediated endothelial cell death.

An Early Developmental Role for miRNAs in the Maintenance of Extraembryonic Stem Cells in the Mouse Embryo

The two first cell fate decisions taken in the mammalian embryo generate three distinct cell lineages: one embryonic, the epiblast, and two extraembryonic, the trophoblast and primitive endoderm. miRNAs are essential for early development, but it is not known if they are utilized in the same way in these three lineages. We find that in the pluripotent epiblast they inhibit apoptosis by blocking the expression of the proapoptotic protein Bcl2l11 (Bim) but play little role in the initiation of gastrulation. In contrast, in the trophectoderm, miRNAs maintain the trophoblast stem cell compartment by directly inhibiting expression of Cdkn1a (p21) and Cdkn1c (p57), and in the primitive endoderm, they prevent differentiation by maintaining ERK1/2 phosphorylation through blocking the expression of Mapk inhibitors. Therefore, we show that there are fundamental differences in how stem cells maintain their developmental potential in embryonic and extraembryonic tissues through miRNAs.

Evolution of the mammalian embryonic pluripotency gene regulatory network

Embryonic pluripotency in the mouse is established and maintained by a gene-regulatory network under the control of a core set of transcription factors that include octamer-binding protein 4 (Oct4; official name POU domain, class 5, transcription factor 1, Pou5f1), sex-determining region Y (SRY)-box containing gene 2 (Sox2), and homeobox protein Nanog. Although this network is largely conserved in eutherian mammals, very little information is available regarding its evolutionary conservation in other vertebrates. We have compared the embryonic pluripotency networks in mouse and chick by means of expression analysis in the pregastrulation chicken embryo, genomic comparisons, and functional assays of pluripotency-related regulatory elements in ES cells and blastocysts. We find that multiple components of the network are either novel to mammals or have acquired novel expression domains in early developmental stages of the mouse. We also find that the downstream action of the mouse core pluripotency factors is mediated largely by genomic sequence elements nonconserved with chick. In the case of Sox2 and Fgf4, we find that elements driving expression in embryonic pluripotent cells have evolved by a small number of nucleotide changes that create novel binding sites for core factors. Our results show that the network in charge of embryonic pluripotency is an evolutionary novelty of mammals that is related to the comparatively extended period during which mammalian embryonic cells need to be maintained in an undetermined state before engaging in early differentiation events.

MicroRNAs control the apoptotic threshold in primed pluripotent stem cells through regulation of BIM

Mammalian primed pluripotent stem cells have been shown to be highly susceptible to cell death stimuli due to their low apoptotic threshold, but how this threshold is regulated remains largely unknown. Here we identify microRNA (miRNA)-mediated regulation as a key mechanism controlling apoptosis in the post-implantation epiblast. Moreover, we found that three miRNA families, miR-20, miR-92, and miR-302, control the mitochondrial apoptotic machinery by fine-tuning the levels of expression of the proapoptotic protein BIM. These families therefore represent an essential buffer needed to maintain cell survival in stem cells that are primed for not only differentiation but also cell death.

Crosstalk between Nodal/activin and MAPK p38 signaling is essential for anterior-posterior axis specification.

Summary Nodal/activin signaling plays a key role in anterior-posterior (A-P) axis formation by inducing the anterior visceral endoderm (AVE), the extraembryonic signaling center that initiates anterior patterning in the embryo. Here we provide direct evidence that the mitogen-activated protein kinase (MAPK) p38 regulates AVE specification through a crosstalk with the Nodal/activin signaling pathway. We show that p38 activation is directly stimulated by Nodal/activin and fails to be maintained upon inhibition of this pathway both in vivo and in vitro. In turn, p38 strengthens the Nodal signaling response by phosphorylating the Smad2 linker region and enhancing the level of Smad2 activation. Furthermore, we demonstrate that this p38 amplification loop is essential for correct specification of the AVE in two ways: first, by showing that inhibiting p38 activity in 5.5 days postcoitum embryo cultures leads to a switch from AVE to an extraembryonic visceral endoderm cell identity, and second, by demonstrating that genetically reducing p38 activity in a Nodal-sensitive background leads to a failure of AVE specification in vivo. Collectively, our results reveal a novel role for p38 in regulating the threshold of Nodal signaling and propose a new mechanism by which A-P axis development can be reinforced during early embryogenesis.

Evolutionarily conserved A-to-I editing increases protein stability of the alternative splicing factor Nova1

The structural complexity of the vertebrate brain is mirrored by its unparalleled transcriptome complexity. In particular, two post-transcriptional processes, alternative splicing and RNA editing, greatly diversify brain transcriptomes. Here we report a close connection between these two processes: we show A-to-I RNA editing in Nova1, a key brain-specific regulator of alternative splicing. Nova1 editing levels increase during embryonic development in mouse and chicken brains and show significant variation across postnatal brain regions. Evolutionary conservation of both editing and editing-associated RNA secondary structure of the Nova1 mRNA for 300 million years attests to the functional importance of Nova1 editing. Using a combination of different assays in human HEK293T cell lines, we report a novel post-translational role for this RNA editing. Whereas functional assays showed no effect of RNA editing on the regulatory splicing activity of the encoded proteins, we found evidence that edited forms exhibit reduced proteasome targeting and increased protein half-life. In addition, we found evidence for similar regulation of protein half-life by an evolutionarily conserved alternative splicing event in Nova1. These results open new venues of research on the multi-level integration of gene expression by: (1) revealing the novel role of RNA editing in regulating protein stability, and (2) establishing protein stability as a new target of multifaceted regulation.

Understanding the regulatory genome.

The sequencing of the whole genome of multiple species provides us with the instruction book of how to build an organism and make it work, plus a detailed history of how diversity was generated during evolution. Unfortunately, we still understand only a small fraction, which is locating where genes are and deciphering the proteins they code for. The next step is to understand how the correct amount of gene products are produced in space and time to obtain a fully functioning organism, from the egg to the adult. This is what is known as the regulatory genome, a term coined by Eric H. Davidson. In this review, we examine what we know about gene regulation from a genomic point of view, revise the current in silico, in vitro and in vivo methodological approaches to study transcriptional regulation, and point to the power of phylogenetic footprinting as a guide to regulatory element discovery. The advantages and limitations of each approach are considered, with the emerging view that only large-scale studies and data-crunching will give us insight into the language of genomic regulatory systems, and allow the discovery of regulatory codes in the genome.

MiRNA-mediated regulation of cell signaling and homeostasis in the early mouse embryo.

At the time of implantation the mouse embryo is composed of three tissues the epiblast, trophectoderm and primitive endoderm. As development progresses the epiblast goes on to form the foetus whilst the trophectoderm and primitive endoderm give rise to extra-embryonic structures with important roles in embryo patterning and nutrition. Dramatic changes in gene expression occur during early embryo development and these require regulation at different levels. miRNAs are small non coding RNAs that have emerged over the last decade as important post-transcriptional repressors of gene expression. The roles played by miRNAs during early mammalian development are only starting to be elucidated. In order to gain insight into the function of miRNAs in the different lineages of the early mouse embryo we have analysed in depth the phenotype of embryos and extra-embryonic stem cells mutant for the miRNA maturation protein Dicer. This study revealed that miRNAs are involved in regulating cell signaling and homeostasis in the early embryo. Specifically, we identified a role for miRNAs in regulating the Erk signaling pathway in the extra-embryonic endoderm, cell cycle progression in extra-embryonic tissues and apoptosis in the epiblast.

Comparison of extraembryonic expression of Eomes and Cdx2 in pregastrulation chick and mouse embryo unveils regulatory changes along evolution

In the mouse blastocyst, Eomes and Cdx2 are critical for establishing the trophoectoderm, the precursor of the placenta. To better understand how the trophoectoderm lineage arose in mammals during evolution, we examined the expression of their orthologues in the pregastrulation chick embryo and found that, while both genes are expressed in extraembryonic tissues, their temporal pattern of expression differs from what occurs in mouse. Moreover, we failed to detect expression of other genes specific from the mouse trophoectoderm in extraembryonic regions of the chick. Also unlike the mouse, chick Eomes is expressed in primordial germ cells. Finally, conserved noncoding elements in the Eomes genomic region are unable to drive trophoectoderm restricted expression in the mouse blastocyst, but do so in conserved sites of expression such as the forebrain. These results suggest that critical changes in the gene regulatory networks controlling extraembryonic development accompanied the appearance of the trophoectoderm in mammals. Developmental Dynamics 239:620–629, 2010.