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

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]

Amphilimus- vs. zotarolimus-eluting stents in patients with diabetes mellitus and coronary artery disease: the SUGAR trial

Aim: Patients with diabetes mellitus are at high risk of adverse events after percutaneous revascularization, with no differences in outcomes between most contemporary drug-eluting stents. The Cre8 EVO stent releases a formulation of sirolimus with an amphiphilic carrier from laser-dug wells, and has shown clinical benefits in diabetes. We aimed to compare Cre8 EVO stents to Resolute Onyx stents (a contemporary polymer-based zotarolimus-eluting stent) in patients with diabetes. Methods and results: We did an investigator-initiated, randomized, controlled, assessor-blinded trial at 23 sites in Spain. Eligible patients had diabetes and required percutaneous coronary intervention. A total of 1175 patients were randomly assigned (1:1) to receive Cre8 EVO or Resolute Onyx stents. The primary endpoint was target-lesion failure, defined as a composite of cardiac death, target-vessel myocardial infarction, and clinically indicated target-lesion revascularization at 1-year follow-up. The trial had a non-inferiority design with a 4% margin for the primary endpoint. A superiority analysis was planned if non-inferiority was confirmed. There were 106 primary events, 42 (7.2%) in the Cre8 EVO group and 64 (10.9%) in the Resolute Onyx group [hazard ratio (HR) 0.65, 95% confidence interval (CI) 0.44 to 0.96; pnon-inferiority <0.001; psuperiority = 0.030]. Among the secondary endpoints, Cre8 EVO stents had significantly lower rate than Resolute Onyx stents of target-vessel failure (7.5% vs 11.1%, HR 0.67, 95% CI 0.46 to 0.99; p = 0.042). Probable or definite stent thrombosis and all-cause death were not significantly different between groups. Conclusions: In patients with diabetes, Cre8 EVO stents were non-inferior to Resolute Onyx stents with regard to target-lesion failure composite outcome. An exploratory analysis for superiority at 1 year suggests that the Cre8 EVO stents might be superior to Resolute Onyx stents with regard to the same outcome

Amphilimus- vs. zotarolimus-eluting stents in patients with diabetes mellitus and coronary artery disease: the SUGAR trial.

AIM: Patients with diabetes mellitus are at high risk of adverse events after percutaneous revascularization, with no differences in outcomes between most contemporary drug-eluting stents. The Cre8 EVO stent releases a formulation of sirolimus with an amphiphilic carrier from laser-dug wells, and has shown clinical benefits in diabetes. We aimed to compare Cre8 EVO stents to Resolute Onyx stents (a contemporary polymer-based zotarolimus-eluting stent) in patients with diabetes. METHODS AND RESULTS: We did an investigator-initiated, randomized, controlled, assessor-blinded trial at 23 sites in Spain. Eligible patients had diabetes and required percutaneous coronary intervention. A total of 1175 patients were randomly assigned (1:1) to receive Cre8 EVO or Resolute Onyx stents. The primary endpoint was target-lesion failure, defined as a composite of cardiac death, target-vessel myocardial infarction, and clinically indicated target-lesion revascularization at 1-year follow-up. The trial had a non-inferiority design with a 4% margin for the primary endpoint. A superiority analysis was planned if non-inferiority was confirmed. There were 106 primary events, 42 (7.2%) in the Cre8 EVO group and 64 (10.9%) in the Resolute Onyx group [hazard ratio (HR): 0.65, 95% confidence interval (CI): 0.44-0.96; Pnon-inferiority < 0.001; Psuperiority = 0.030]. Among the secondary endpoints, Cre8 EVO stents had significantly lower rate than Resolute Onyx stents of target-vessel failure (7.5% vs. 11.1%, HR: 0.67, 95% CI: 0.46-0.99; P = 0.042). Probable or definite stent thrombosis and all-cause death were not significantly different between groups. CONCLUSION: In patients with diabetes, Cre8 EVO stents were non-inferior to Resolute Onyx stents with regard to target-lesion failure composite outcome. An exploratory analysis for superiority at 1 year suggests that the Cre8 EVO stents might be superior to Resolute Onyx stents with regard to the same outcome. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov: NCT03321032

Onset of cell differentiation and first lineages decisions in the mouse embryo through the Notch pathway

A central question in developmental biology is how a single cell, the zygote, divides and differentiates to generate all the specialised cells that will build a whole organism. The zygote has totipotent capacity, which means that it is able to give rise to any necessary cell type (embryonic or extraembryonic) to form an adult individual. This capacity is gradually reduced during embryonic development, as cells make fate decisions that increase their specialisation at the expense of restricting their developmental potential. The first lineage choice of the mammalian embryo occurs before its implantation in the maternal uterus (so-called preimplantation development) at the blastocyst stage, and leads to the appearance of the first morphologically distinct cell populations: the trophectoderm and the inner cell mass. The trophectoderm is characteristic of mammals and will give rise to extraembryonic tissues such as the placenta, while the inner cell mass will form the embryo proper. Cdx2 is the key gene required for the specification of the trophectoderm and is regulated by the cooperation of two signalling pathways: Hippo and Notch. The Hippo pathway functions as a readout of intracellular polarity cues starting at the morula stage, but little is known about the role of Notch in preimplantation before the blastocyst stage. By using genetic and pharmacological tools in vivo, together with image analysis of single embryos, we have found an early requirement for Notch, which is active from the 4-cell stage, and precedes that of Hippo in the regulation of Cdx2. Moreover, transcriptomic analysis identified novel Notch targets at these stages including early naïve pluripotency markers or transcriptional repressors such as Tle4. Our results unveil a role for Notch in driving the transition towards a more committed state during the gradual loss of potency that takes place in the early mouse embryo prior to the first lineage decisions.S

Onset of cell differentiation and first lineage decisions in the mouse embryo through the notch pathway

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Medicina, Departamento de Bioquímica. Fecha de lectura: 25-01-2019Esta tesis tiene embargado el acceso al texto completo hasta el 25-07-2020A central question in developmental biology is how a single cell, the zygote, divides and differentiates to generate all the specialised cells that will build a whole organism. The zygote has totipotent capacity, which means that it is able to give rise to any necessary cell type (embryonic or extraembryonic) to form an adult individual. This capacity is gradually reduced during embryonic development, as cells make fate decisions that increase their specialisation at the expense of restricting their developmental potential. The first lineage choice of the mammalian embryo occurs before its implantation in the maternal uterus (so-called preimplantation development) at the blastocyst stage, and leads to the appearance of the first morphologically distinct cell populations: the trophectoderm and the inner cell mass. The trophectoderm is characteristic of mammals and will give rise to extraembryonic tissues such as the placenta, while the inner cell mass will form the embryo proper. Cdx2 is the key gene required for the specification of the trophectoderm and is regulated by the cooperation of two signalling pathways: Hippo and Notch. The Hippo pathway functions as a readout of intracellular polarity cues starting at the morula stage, but little is known about the role of Notch in preimplantation before the blastocyst stage. By using genetic and pharmacological tools in vivo, together with image analysis of single embryos, we have found an early requirement for Notch, which is active from the 4-cell stage, and precedes that of Hippo in the regulation of Cdx2. Moreover, transcriptomic analysis identified novel Notch targets at these stages including early naïve pluripotency markers or transcriptional repressors such as Tle4. Our results unveil a role for Notch in driving the transition towards a more committed state during the gradual loss of potency that takes place in the early mouse embryo prior to the first lineage decisions.Una pregunta central en el campo de la biología del desarrollo es cómo una única célula, el zigoto, se divide y se diferencia para generar todas las células especializadas que forman un organismo. El zigoto tiene una capacidad totipotente, es decir, es capaz de dar lugar a cualquier tipo celular (embrionario o extraembrionario) necesario para la formación de un individuo adulto. Esta capacidad se reduce gradualmente durante el desarrollo embrionario dado que las células toman decisiones de linaje que suponen un aumento de su especialización a expensas de un potencial más restringido. La primera decisión de linaje del embrión de mamíferos ocurre antes de su implantación en el útero materno (a lo que se llama desarrollo preimplantacional) en el estadio de blastocisto, y supone la aparición de las primeras poblaciones celulares morfológicamente distintas: el trofoectodermo y la masa celular interna. El trofoectodermo es característico de los mamíferos y dará lugar a estructuras extraembrionarias como la placenta, mientras que la masa celular interna formará el embrión. Cdx2 es un gen clave necesario para la especificación del trofoectodermo, y se regula gracias a la cooperación de dos vías de señalización: Hippo y Notch. La vía de Hippo traduce las señales que recibe de polaridad intracelular desde el estadio de mórula, pero el papel de la vía de Notch en estadios preimplantacionales es poco conocido. Mediante el empleo de herramientas genéticas y farmacológicas in vivo, junto con análisis de imagen de embriones individuales, hemos descubierto un requerimiento temprano de la vía de Notch, que está activa desde el estadio de 4 células, precediendo así al de la vía de Hippo en la regulación de Cdx2. Además, análisis transcriptómicos nos han llevado a identificar nuevas dianas de Notch en estos estadios, entre los que se incluyen marcadores de pluripotencia naíf o represores transcripcionales como Tle4. Nuestros resultados revelan, por tanto, que Notch tiene un papel dirigiendo la transición hacia estados más comprometidos celularmente durante la pérdida gradual de potencial que tiene lugar en el embrión temprano de ratón, antes de la primera decisión de linaje

Our First Choice: Cellular and Genetic Underpinnings of Trophectoderm Identity and Differentiation in the Mammalian Embryo.

The trophectoderm (TE) is the first cell population to appear in the mammalian preimplantation embryo, as the result of the differentiation of totipotent blastomeres located on the outer surface of the late morula. Trophectodermal cells arrange in a monolayer covering the expanding blastocyst and acquire an epithelial phenotype with distinct apicobasal polarity and a basal lamina placed toward the blastocyst interior. During later development through the periimplantation and gastrulation stages, the TE gives rise to extraembryonic membranes and cell types that will eventually form most of the fetal placenta, the specialized organ through which the embryo obtains maternal nourishment necessary for subsequent exponential growth. The specification of the TE is controlled by the combination of morphological cues arising from cell polarity with differential activity of signaling pathways such as Hippo and Notch, and the restriction to outer cells of lineage specifiers such as CDX2. This is possibly the first symmetry-breaking decision undertaken by the uncommitted cells produced by a handful of mitosis divisions from the newly fertilized zygote. Understanding how this cell lineage is specified will therefore provide unique information about development, differentiation, and how the interplay between cellular morphology and signaling and regulatory factors results in a correctly 3D-patterned embryo.Work in our lab is supported by the Spanish government (FPI-SO Fellowship to S.M., FPI Fellowship to J.G.S.A., and grant BFU2014-54608-P to M.M.). The CNIC is supported by the Ministry of Economy, Industry and Competitiveness (MEIC) and the Pro CNIC Foundation, and is a Severo Ochoa Center of Excellence (SEV-2015-0505).S

Signaling pathways in mammalian preimplantation development: Linking cellular phenotypes to lineage decisions.

The first stages of mammalian development, before implantation of the embryo in the maternal uterus, result in the establishment of three cell populations in the blastocyst: trophectoderm, epiblast, and primitive endoderm. These events involve only a small number of cells, and are initiated by morphological differences among them related to cell adhesion and polarity. Much attention has been paid to the master transcription factors that are critical for establishing and maintaining early lineage choices. Nevertheless, a large body of work also reveals that additional molecular mechanisms are involved. Here, we provide an updated view of the role of different signaling pathways in the first stages of mouse development, and how their cross-talk and interplay determine the initial lineage decisions occurring in the blastocyst. We will also discuss how these pathways are critical for translating cellular phenotypes, the product of the morphogenetic events occurring at these stages, into transcriptional responses and expression of lineage-specifying transcription factors. Developmental Dynamics 246:245-261, 2017. © 2016 Wiley Periodicals, Inc.Grant sponsor: Spanish government; Grant number: BFU2014-54608-P; Grant sponsor: CNIC (supported by the Spanish Ministerio de Economia y Competitividad and the Pro-CNIC Foundation, and is a Severo Ochoa Center of Excellence); SEV-2015-0505.S

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