The pluripotency factor NANOG controls primitive hematopoiesis and directly regulates Tal1

Progenitors of the first hematopoietic cells in the mouse arise in the early embryo from Brachyury-positive multipotent cells in the posterior-proximal region of the epiblast, but the mechanisms that specify primitive blood cells are still largely unknown. Pluripotency factors maintain uncommitted cells of the blastocyst and embryonic stem cells in the pluripotent state. However, little is known about the role played by these factors during later development, despite being expressed in the postimplantation epiblast. Using a dual transgene system for controlled expression at postimplantation stages, we found that Nanog blocks primitive hematopoiesis in the gastrulating embryo, resulting in a loss of red blood cells and downregulation of erythropoietic genes. Accordingly, Nanog-deficient embryonic stem cells are prone to erythropoietic differentiation. Moreover, Nanog expression in adults prevents the maturation of erythroid cells. By analysis of previous data for NANOG binding during stem cell differentiation and CRISPR/Cas9 genome editing, we found that Tal1 is a direct NANOG target. Our results show that Nanog regulates primitive hematopoiesis by directly repressing critical erythroid lineage specifiers.This work was supported by the Spanish government (grant BFU2014-54608-P and BFU2017-84914-P to MM; grants RYC-2011-09209 and BFU-2012-35892 to JI). The Gottgens and Nichols laboratories are supported by core funding from the Wellcome Trust and MRC to the Wellcome and MRC Cambridge Stem Cell Institute. The CNIC is supported by the Spanish Ministry of Science, Innovation and Universities (MINECO) and the Pro CNIC Foundation, and is a Severo Ochoa Center of Excellence (SEV-2015-0505)S

Pluripotency factors regulate the onset of Hox cluster activation in the early embryo

Pluripotent cells are a transient population of the mammalian embryo dependent on transcription factors, such as OCT4 and NANOG, which maintain pluripotency while suppressing lineage specification. However, these factors are also expressed during early phases of differentiation, and their role in the transition from pluripotency to lineage specification is largely unknown. We found that pluripotency factors play a dual role in regulating key lineage specifiers, initially repressing their expression and later being required for their proper activation. We show that Oct4 is necessary for activation of HoxB genes during differentiation of embryonic stem cells and in the embryo. In addition, we show that the HoxB cluster is coordinately regulated by OCT4 binding sites located at the 3′ end of the cluster. Our results show that core pluripotency factors are not limited to maintaining the precommitted epiblast but are also necessary for the proper deployment of subsequent developmental programs

Nanog regulates Pou3f1 expression at the exit from pluripotency during gastrulation.

Pluripotency is regulated by a network of transcription factors that maintain early embryonic cells in an undifferentiated state while allowing them to proliferate. NANOG is a critical factor for maintaining pluripotency and its role in primordial germ cell differentiation has been well described. However, Nanog is expressed during gastrulation across all the posterior epiblast, and only later in development is its expression restricted to primordial germ cells. In this work, we unveiled a previously unknown mechanism by which Nanog specifically represses genes involved in anterior epiblast lineage. Analysis of transcriptional data from both embryonic stem cells and gastrulating mouse embryos revealed Pou3f1 expression to be negatively correlated with that of Nanog during the early stages of differentiation. We have functionally demonstrated Pou3f1 to be a direct target of NANOG by using a dual transgene system for the controlled expression of Nanog Use of Nanog null ES cells further demonstrated a role for Nanog in repressing a subset of anterior neural genes. Deletion of a NANOG binding site (BS) located nine kilobases downstream of the transcription start site of Pou3f1 revealed this BS to have a specific role in the regionalization of the expression of this gene in the embryo. Our results indicate an active role of Nanog inhibiting neural regulatory networks by repressing Pou3f1 at the onset of gastrulation.This article has an associated First Person interview with the joint first authors of the paper.This work was funded by the Spanish government [grant BFU2017-84914-P to M.M.]. The Gottgens laboratory is supported by core funding from the Wellcome Trust and Medical Research Council to the Wellcome and Medical Research Council Cambridge Stem Cell Institute. The CNIC is supported by the Instituto de Salud Carlos III (ISCIII), the Ministerio de Ciencia, Innovación y Universidades (MCNU) and the Pro CNIC Foundation, and is a Severo Ochoa Center of Excellence [SEV-2015-0505]

Transitions in cell potency during early mouse development are driven by Notch

The Notch signalling pathway plays fundamental roles in diverse developmental processes in metazoans, where it is important in driving cell fate and directing differentiation of various cell types. However, we still have limited knowledge about the role of Notch in early preimplantation stages of mammalian development, or how it interacts with other signalling pathways active at these stages such as Hippo. By using genetic and pharmacological tools in vivo, together with image analysis of single embryos and pluripotent cell culture, we have found that Notch is active from the 4-cell stage. Transcriptomic analysis in single morula identified novel Notch targets, such as early naïve pluripotency markers or transcriptional repressors such as TLE4. Our results reveal a previously undescribed role for Notch in driving transitions during the gradual loss of potency that takes place in the early mouse embryo prior to the first lineage decisions.This work was supported by the Spanish government (grants BFU2017-84914-P and BFU2015-72319-EXP to MM; FPI-SO Fellowship to SM); and grants NIH-R01DK084391, NIH-R01HD094868 and NIH-P30CA008748 to AKH. The CNIC is supported by the Spanish Ministry of Science, Innovation and Universities and the Pro CNIC Foundation, and is a Severo Ochoa Center of Excellence (SEV-2015-0505).S

Nanog regulates Pou3f1 expression at the exit from pluripotency during gastrulation

Pluripotency is regulated by a network of transcription factors that maintain early embryonic cells in an undifferentiated state while allowing them to proliferate. NANOG is a critical factor for maintaining pluripotency and its role in primordial germ cell differentiation has been well described. However, Nanog is expressed during gastrulation across all the posterior epiblast, and only later in development is its expression restricted to primordial germ cells. In this work, we unveiled a previously unknown mechanism by which Nanog specifically represses genes involved in anterior epiblast lineage. Analysis of transcriptional data from both embryonic stem cells and gastrulating mouse embryos revealed Pou3f1 expression to be negatively correlated with that of Nanog during the early stages of differentiation. We have functionally demonstrated Pou3f1 to be a direct target of NANOG by using a dual transgene system for the controlled expression of Nanog Use of Nanog null ES cells further demonstrated a role for Nanog in repressing a subset of anterior neural genes. Deletion of a NANOG binding site (BS) located nine kilobases downstream of the transcription start site of Pou3f1 revealed this BS to have a specific role in the regionalization of the expression of this gene in the embryo. Our results indicate an active role of Nanog inhibiting neural regulatory networks by repressing Pou3f1 at the onset of gastrulation.This article has an associated First Person interview with the joint first authors of the paper.This work was funded by the Spanish government [grant BFU2017-84914-P to M.M.]. The Gottgens laboratory is supported by core funding from the Wellcome Trust and Medical Research Council to theWellcome and Medical Research Council Cambridge Stem Cell Institute. The CNIC is supported by the Instituto de Salud Carlos III (ISCIII), the Ministerio de Ciencia, Innovación y Universidades MCNU) and the Pro CNIC Foundation, and is a Severo Ochoa Center of Excellence [SEV-2015-0505].S

Long-range regulatory interactions at the 4q25 atrial fibrillation risk locus involve PITX2c and ENPEP

Background: Recent genome-wide association studies have uncovered genomic loci that underlie an increased risk for atrial fibrillation, the major cardiac arrhythmia in humans. The most significant locus is located in a gene desert at 4q25, approximately 170 kilobases upstream of PITX2, which codes for a transcription factor involved in embryonic left-right asymmetry and cardiac development. However, how this genomic region functionally and structurally relates to PITX2 and atrial fibrillation is unknown. Results: To characterise its function, we tested genomic fragments from 4q25 for transcriptional activity in a mouse atrial cardiomyocyte cell line and in transgenic mouse embryos, identifying a non-tissue-specific potentiator regulatory element. Chromosome conformation capture revealed that this region physically interacts with the promoter of the cardiac specific isoform of Pitx2. Surprisingly, this regulatory region also interacts with the promoter of the next neighbouring gene, Enpep, which we show to be expressed in regions of the developing mouse heart essential for cardiac electrical activity. Conclusions: Our data suggest that de-regulation of both PITX2 and ENPEP could contribute to an increased risk of atrial fibrillation in carriers of disease-associated variants, and show the challenges that we face in the functional analysis of genome-wide disease associations.We thank Miguel Torres and members of the Manzanares lab for support and comments; Christine Mummery, Jose Luis de la Pompa and Joaquin Rodriguez-Leon for reagents; the CNIC Transgenic Unit for generation of embryos; Stuart Pocock for statistical advice; and Simon Bartlett for English editing. This study was funded by the CNIC Translational Grant Programme (CNIC-08-2009 to MM and DF), the Spanish Ministerio de Economia y Competitividad (grants BFU2011-23083 to MM, BFU2013-41322-P to JLGS, BFU2012-38111 to AA, and CSD2007-00008 to JLGS and MM), the Comunidad Autonoma de Madrid (grant CELLDD-CM to MM), and the Andalusian Government (grant BIO-396 to JLGS). The CNIC is supported by the Spanish Ministerio de Economia y Competitividad and the Pro-CNIC Foundation.S

The pluripotency factor NANOG controls primitive hematopoiesis and directly regulates Tal1.

Progenitors of the first hematopoietic cells in the mouse arise in the early embryo from Brachyury-positive multipotent cells in the posterior-proximal region of the epiblast, but the mechanisms that specify primitive blood cells are still largely unknown. Pluripotency factors maintain uncommitted cells of the blastocyst and embryonic stem cells in the pluripotent state. However, little is known about the role played by these factors during later development, despite being expressed in the postimplantation epiblast. Using a dual transgene system for controlled expression at postimplantation stages, we found that Nanog blocks primitive hematopoiesis in the gastrulating embryo, resulting in a loss of red blood cells and downregulation of erythropoietic genes. Accordingly, Nanog-deficient embryonic stem cells are prone to erythropoietic differentiation. Moreover, Nanog expression in adults prevents the maturation of erythroid cells. By analysis of previous data for NANOG binding during stem cell differentiation and CRISPR/Cas9 genome editing, we found that Tal1 is a direct NANOG target. Our results show that Nanog regulates primitive hematopoiesis by directly repressing critical erythroid lineage specifiers.Wellcome Trust, Bloodwise, Spanish government, Pro CNIC Foundation, Severo Ochoa Center of Excellenc

CTCF counter-regulates cardiomyocyte development and maturation programs in the embryonic heart

Cardiac progenitors are specified early in development and progressively differentiate and mature into fully functional cardiomyocytes. This process is controlled by an extensively studied transcriptional program. However, the regulatory events coordinating the progression of such program from development to maturation are largely unknown. Here, we show that the genome organizer CTCF is essential for cardiogenesis and that it mediates genomic interactions to coordinate cardiomyocyte differentiation and maturation in the developing heart. Inactivation of Ctcf in cardiac progenitor cells and their derivatives in vivo during development caused severe cardiac defects and death at embryonic day 12.5. Genome wide expression analysis in Ctcf mutant hearts revealed that genes controlling mitochondrial function and protein production, required for cardiomyocyte maturation, were upregulated. However, mitochondria from mutant cardiomyocytes do not mature properly. In contrast, multiple development regulatory genes near predicted heart enhancers, including genes in the IrxA cluster, were downregulated in Ctcf mutants, suggesting that CTCF promotes cardiomyocyte differentiation by facilitating enhancer-promoter interactions. Accordingly, loss of CTCF disrupts gene expression and chromatin interactions as shown by chromatin conformation capture followed by deep sequencing. Furthermore, CRISPR-mediated deletion of an intergenic CTCF site within the IrxA cluster alters gene expression in the developing heart. Thus, CTCF mediates local regulatory interactions to coordinate transcriptional programs controlling transitions in morphology and function during heart development

Transcriptional changes in <i>Ctcf</i> mutant hearts.

<p>(A) Iris plot showing enriched GO terms in differentially expressed genes between control and mutant E10.5 hearts (outside circle), together with changes in expression of individual genes (inner circle; red, upregulated in mutants; blue, downregulated). (B) Genes that are up- or downregulated in <i>Ctcf</i> mutant hearts are more likely to have a CTCF binding site in their vicinity (10 or 20 kb surrounding the transcriptional start site) than genes that are expressed in the embryonic heart but do not change. However, only downregulated genes are located closer to heart-specific enhancers. Frequency is expressed relative to that of expressed but unchanged genes. *, p<0.01; **, p<0.0005; Mann-Whitney test. (C) Mean-plots of the distribution of CTCF binding, as determined by ChIP-seq [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006985#pgen.1006985.ref010" target="_blank">10</a>], relative to the TSS (0 on the x-axis) of different groups of genes (legend on the right). Note that the peak of the distributions is located slightly upstream of the TSS, indicative of binding to proximal promoter sequences.</p

Morphological defects in <i>Ctcf</i> mutant embryonic hearts.

<p>Whole mount control and mutant (<i>Ctcf</i>^{<i>fl</i>/<i>fl</i>};<i>Nkx2-5-Cre</i>) embryos at E10.5 (A, B), E11.5 (C, D) and E12.5 (E, F). Arrows in (E) point to the pericardial edema present in mutant embryos. Higher magnification of the heart show morphological defects in <i>Ctcf</i> mutant hearts becoming manifest at E12.5 (F). (G-L) Sections from control and mutant embryos stained with hematoxylin and eosin; higher magnifications (black dashed boxes) for each are shown on the right of each section. Disorganization of the interventricular septum and thinning of the myocardial wall is apparent in <i>Ctcf</i> mutant hearts at E11.5. A, atria; V, ventricle; RA, right atria; LA, left atria; RV, right ventricle; LV, left ventricle; AVC, atrioventricular canal; IVS, interventricular septum. Scale bars, 750 μm (A), 200 μm (B, D), 1 mm (C), 2 mm (E), 800 μm (F), 100 μm (G, H), 200 μm (I-L), and 100 μm in all higher magnifications.</p