The first cell differentiation in mammalian embryos segregates polarized trophectoderm cells from an apolar inner cell mass (ICM). This lineage decision is specified in compacted morulae by cell polarization and adhesion acting on the Yes-associated protein in the Hippo signaling pathway, but the regulatory mechanisms are unclear. We show that morula compaction and ICM formation depend on PC7 and the related proprotein convertases (PCs) Furin and Pace4 and that these proteases jointly regulate cell cell adhesion mediated by E-cadherin processing. We also mapped the spatiotemporal activity profiles of these proteases by live imaging of a transgenic reporter substrate in wild-type and PC mutant embryos. Differential inhibition by a common inhibitor revealed that all three PCs are active in inner and outer cells, but in partially nonoverlapping compartments. E-cadherin processing by multiple PCs emerges as a novel mechanism to modulate cell-cell adhesion and fate allocation.

Endogenous retroviruses (ERVs) undergo de novo DNA methylation during the first few days of mammalian embryogenesis, although the factors that control the targeting of this process are largely unknown. We asked whether KAP1 (KRAB-associated protein 1) is involved in this mechanism because of its previously defined role in maintaining the silencing of ERVs through the histone methyltransferase ESET and histone H3 lysine 9 trimethylation. Here, we demonstrate that introduced ERV sequences are sufficient to direct rapid de novo methylation of a flanked promoter in embryonic stem (ES) cells. This mechanism requires the presence of an ERV sequence-recognizing KRAB zinc-finger protein (ZFP) and both KAP1 and ESET. Furthermore, this process can also take place on a strong cellular promoter and leads to methylation signatures that are subsequently maintained in vivo throughout embryogenesis. Finally, we show that methylation of ERVs residing in the genome is affected by knockout of KAP1 in early embryos. KRAB-ZFPs, KAP1 and ESET are thus likely to be responsible for the early embryonic instatement of stable epigenetic marks at ERV-containing loci.

The fate of pluripotent cells in early mouse embryos is controlled by graded Nodal signals that are activated by the endoproteases Furin and Pace4. Soluble forms of Furin and Pace4 cleave proNodal in vitro and after secretion in transfected cells, but direct evidence for paracrine activity in vivo is elusive. Here, we show that Furin and Pace4 are released by the extraembryonic microenvironment, and that they cleave a membrane-bound reporter substrate in adjacent epiblast cells and activate Nodal to maintain pluripotency. Secreted Pace4 and Furin also stimulated mesoderm formation, whereas endoderm was only induced by Pace4, correlating with a difference in the spatiotemporal distribution of these proteolytic activities. Our analysis of paracrine Furin and Pace4 activities and their in vivo functions significantly advances our understanding of how the epiblast is patterned by its microenvironment. Adding cell-cell communication to the pleiotropic portfolio of these proteases provides a new framework to study proprotein processing also in other relevant contexts.

Axis formation and allocation of pluripotent progenitor cells to the germ layers are governed by the TGF-β-related Nodal precursor and its secreted proprotein convertases (PCs) Furin and Pace4. However, when and where Furin and Pace4 first become active have not been determined. To study the distribution of PCs, we developed a novel cell surface-targeted fluorescent biosensor (cell surface-linked indicator of proteolysis [CLIP]). Live imaging of CLIP in wild-type and Furin- and Pace4-deficient embryonic stem cells and embryos revealed that Furin and Pace4 are already active at the blastocyst stage in the inner cell mass and can cleave membrane-bound substrate both cell autonomously and nonautonomously. CLIP was also cleaved in the epiblast of implanted embryos, in part by a novel activity in the uterus that is independent of zygotic Furin and Pace4, suggesting a role for maternal PCs during embryonic development. The unprecedented sensitivity and spatial resolution of CLIP opens exciting new possibilities to elucidate PC functions in vivo.

Endogenous retroviruses in embryonic stem cells Almost half of the mammalian genome is derived from virus-like entities called retroelements, many of them the result of the incorporation of endogenous retroviruses. Didier Trono and colleagues use a combination of genetic, functional and biochemical analyses to demonstrate that KRAB-associated protein 1 (KAP1)-mediated epigenetic regulation is involved in the early embryonic silencing of intracisternal A particles (IAPs), a still active and highly polymorphic subset of endogenous retroviruses. This work establishes a role for the KAP1 co-repressor in the control of endogenous retroelements during early embryonic development.

Reference EPFL-CONF-160781doi:10.1016/j.ydbio.2008.05.413View record in Web of Science Record created on 2010-11-30, modified on 2017-05-12

We have identified an ethylnitrosourea (ENU)-induced recessive mouse mutation (Vcc) with a pleiotropic phenotype that includes cardiac, tracheoesophageal, anorectal, anteroposterior patterning defects, exomphalos, hindlimb hypoplasia, a presacral mass, renal and palatal agenesis, and pulmonary hypoplasia. It results from a C470R mutation in the proprotein convertase PCSK5 (PC5/6). Compound mutants (Pcsk5(Vcc/null)) completely recapitulate the Pcsk5(Vcc/Vcc) phenotype, as does an epiblast-specific conditional deletion of Pcsk5. The C470R mutation ablates a disulfide bond in the P domain, and blocks export from the endoplasmic reticulum and proprotein convertase activity. We show that GDF11 is cleaved and activated by PCSK5A, but not by PCSK5A-C470R, and that Gdf11-deficient embryos, in addition to having anteroposterior patterning defects and renal and palatal agenesis, also have a presacral mass, anorectal malformation, and exomphalos. Pcsk5 mutation results in abnormal expression of several paralogous Hox genes (Hoxa, Hoxc, and Hoxd), and of Mnx1 (Hlxb9). These include known Gdf11 targets, and are necessary for caudal embryo development. We identified nonsynonymous mutations in PCSK5 in patients with VACTERL (vertebral, anorectal, cardiac, tracheoesophageal, renal, limb malformation OMIM 192350) and caudal regression syndrome, the phenotypic features of which resemble the mouse mutation. We propose that Pcsk5, at least in part via GDF11, coordinately regulates caudal Hox paralogs, to control anteroposterior patterning, nephrogenesis, skeletal, and anorectal development.

Access the most recent version at doi: 2008 22: 1465-1477 Genes & Dev. Ragoussis, Daniel Constam, Nabil G. Seidah, Annik Prat and Shoumo Bhattacharya Neubauer, Andrew Morris, Steve D. Brown, Charles Shaw-Smith, Armando Cama, Valeria Capra, Jiannis Sebastian J. Arnold, Paul Johnson, Zuzanna Tymowska-Lalanne, Dave Stammers, Kieran Clarke, Stefan Franklyn, Kamel El Omari, Joanna Jefferis, Jamie Bentham, Jennifer M. Taylor, Jurgen E. Schneider, Dorota Szumska, Guido Pieles, Rachid Essalmani, Michal Bilski, Daniel Mesnard, Kulvinder Kaur, Angela Pcsk5 malformations in mice with mutation in the proprotein convertase VACTERL/caudal regression/Currarino syndrome-like

The glycosylphosphatidylinositol (GPI)-anchored proteoglycan Cripto binds Nodal and its type I receptor Alk4 to activate Smad2,3 transcription factors, but a role during Nodal precursor processing has not been described. We show that Cripto also binds the proprotein convertases Furin and PACE4 and localizes Nodal processing at the cell surface. When coexpressed as in early embryonic cells, Cripto and uncleaved Nodal already associated during secretion, and a Cripto-interacting region in the Nodal propeptide potentiated the effect of proteolytic maturation on Nodal signalling. Disruption of the trans-Golgi network (TGN) by brefeldin A blocked secretion, but export of Cripto and Nodal to the cell surface was not inhibited, indicating that Nodal is exposed to extracellular convertases before entering the TGN/endosomal system. Density fractionation and antibody uptake experiments showed that Cripto guides the Nodal precursor in detergent-resistant membranes to endocytic microdomains marked by GFP-Flotillin. We conclude that Nodal processing and endocytosis are coupled in signal-receiving cells.

Sol-gel Zr0.1Ce0.9O2 and Zr0.1(Ce0.75Pr0.25)O2 mixed oxides and coprecipitated pure ceria CeO2 displaying the fluorine type structure have been used as platinum or ruthenium catalysts’ supports for catalytic wet air oxidation (CWAO) of aqueous solution of acetic acid (78 mmol.L-1). These catalysts were prepared by conventional impregnation (5 wt-%) from platinum and ruthenium precursor salts or by exchange (~2 wt-%) in the case of ruthenium. A screening of catalysts in CWAO at 200°C under 2 MPa was performed and reveals that the best platinum catalyst is supported on pure ceria displaying large surface. For ruthenium catalysts, the highest conversion after 3 hours of reaction has been reached by the Ru/Zr-Ce-O system.

Platinum catalysts (2.5 wt%) prepared by impregnation on pure commercial ceria and on Zr-0.1(Ce0.75Pr0.25)(0.9)O-2 sol-gel mixed oxide were synthesized and used for catalytic wet air oxidation (CWAO) of acetic acid. The influence of the platinum sintering (reducing treatment) was studied for both supports to modify the oxygen transfer from the gas phase onto the metallic active site of the catalyst and also to determine the effect of this treatment on the catalytic activity and stability. The catalysts were characterized by BET, TEM, XRD, FT-IR and hydrogen chemisorption before and after CWAO reaction, and oxygen storage capacity (OSC) was measured. The nature of the support [CeO2 and Zr-0.1(Ce0.75Pr0.25)(0.9)O-2 compounds] and the preparation conditions led to samples displaying a Pt dispersion ranging from 0.4 to 41 %, and BET surface area varying from 4 to 45 m(2) g(-1). The results demonstrate poisoning of the catalysts during the CWAO experiments due to the formation of carbonate species. This limits the performance of the mixed oxide-based catalysts displaying the highest OSC values. A correlation was established between the OSC of the catalysts and the formation of carbonate species. Nevertheless, there is no correlation between OSC and catalytic activity, with the ceria-based samples showing the highest reaction rate. An optimal platinum crystallite size is required to achieve the highest conversion of acetic acid in terms of turnover frequency values. (c) 2007 Elsevier Inc. All rights reserved.

Anteroposterior (AP) polarity in the mammalian embryo is specified during gastrulation when naive progenitor cells in the primitive ectoderm are recruited into the primitive streak to form mesoderm and endoderm. At the opposite pole, this process is inhibited by signals previously induced in distal visceral endoderm (DVE). Both DVE and primitive streak formation, and hence positioning of the AP axis, rely on the TGFbeta family member Nodal and its proprotein convertases Furin and Pace4. Here, we show that Nodal and Furin are initially co-expressed in the primitive endoderm together with a subset of DVE markers such as Lefty1 and Hex. However, with the appearance of extra-embryonic ectoderm (ExE), DVE formation is transiently inhibited. During this stage, Nodal activity is essential to specify embryonic VE and restrict the expression of Furin to the extra-embryonic region. Activation of Nodal is also necessary to maintain determinants of pluripotency such as Oct4, Nanog and Foxd3 during implantation, and to stimulate elongation of the egg cylinder, before inducing DVE and germ layer formation. We conclude that Nodal is already activated in primitive endoderm, but induces a functional DVE only after promoting the expansion of embryonic VE and pluripotent progenitor cells in the epiblast.

During early mouse development, the subtilisin-like proprotein convertases (SPC) Furin and PACE4 pattern the primitive ectoderm and visceral endoderm, presumably by activating the TGF beta-related Nodal precursor. Here, mutation of the SPC motif provides direct evidence that Nodal processing is essential to specify anterior visceral endoderm and mesendoderm. Surprisingly, however, the Nodal precursor binds and activates activin receptors to maintain expression of Furin, PACE4, and Bmp4 in extraembryonic ectoderm at a distance from the Nodal source. In return, Bmp4 induces Wnt3, which amplifies Nodal expression in the epiblast and mediates induction of mesoderm. We conclude that uncleaved Nodal sustains the extraembryonic source of proprotein convertases and Bmp4 to amplify Nodal signaling in two nonredundant feedback loops with dual timescales and to localize primitive streak formation at the posterior pole. Based on mathematical modeling, we discuss how these sequential loops control cell fate.

Background: When the anterior-posterior axis of the mouse embryo becomes explicit at gastrulation, it is almost perpendicular to the long uterine axis. This led to the belief that the uterus could play a key role in positioning this future body axis.Results: Here, we demonstrate that when the anterior-posterior axis first emerges it does not respect the axes of the uterus but, rather, the morphology of the embryo. Unexpectedly, the emerging anterior-posterior axis is initially aligned not with the long, but the short axis of the embryo. Then whether the embryo develops in vitro or in utero, the anterior-posterior axis becomes aligned with the long axis of embryo just prior to gastrulation. Of three mechanisms that could account for this apparent shift in anterior-posterior axis orientation-cell migration, spatial change of gene expression, or change in embryo shape-lineage tracing studies favor a shape change accompanied by restriction of the expression domain of anterior markers. This property of the embryo must be modulated by interactions with the uterus as ultimately the anterior-posterior and long axes of the embryo align with the left-right uterine axis. Conclusions: The emerging anterior-posterior axis relates to embryo morphology rather than that of the uterus. The apparent shift in its orientation to align with the long embryonic axis and with the uterus is associated with a change in embryo shape and a refinement of anterior gene expression pattern. This suggests an interdependence between anterior-posterior gene expression, the shape of the embryo, and the uterus.

Ce-Pr-O mixed oxides prepared by coprecipitation and calcined at 1173 K reveal a non stoichiometric structure with oxygen vacancies in the bulk. In situ XRD and Raman spectroscopy were used to investigate these properties simultaneously and identified a thermal expansion coefficient twice as high as that of pure ceria at room temperature and the presence of a Raman band at 560 cm(-1), characteristic of the oxygen vacancies. These results confirm that praseodymium atoms are able to induce oxygen mobility in the bulk at 298 K. This is due to a structure containing a high number of anionic vacancies pre-existing at ambient temperature.

2,5-dimethyl-1,3,4-thiadiazole (1) under bar reacts at low temperature sequentially with lithium diisopropylamide and carbonyl derivatives (2) under bar to prepare, according to the ratio of reagents, either 2-methyl-5-[2-hydroxy-2-arylethyl (or alkyl)]-1,3,4-thiadiazoles (3) under bar or 2,5-bis[2-hydroxy-2-arylethyl (or alkyl)l-l,3,4-thiadiazoles (4) under bar.