Wossidlo M.,Saarland University |
Nakamura T.,Osaka University |
Lepikhov K.,Saarland University |
Marques C.J.,Babraham Institute |
And 7 more authors.
Nature Communications | Year: 2011
The epigenomes of early mammalian embryos are extensively reprogrammed to acquire a totipotent developmental potential. A major initial event in this reprogramming is the active loss/demethylation of 5-methylcytosine (5mC) in the zygote. Here, we report on findings that link this active demethylation to molecular mechanisms. We detect 5-hydroxymethylcytosine (5hmC) as a novel modification in mouse, bovine and rabbit zygotes. On zygotic development 5hmC accumulates in the paternal pronucleus along with a reduction of 5mC. A knockdown of the 5hmC generating dioxygenase Tet3 simultaneously affects the patterns of 5hmC and 5mC in the paternal pronucleus. This finding links the loss of 5mC to its conversion into 5hmC. The maternal pronucleus seems to be largely protected against this mechanism by PGC7/Dppa3/Stella, as in PGC7 knockout zygotes 5mC also becomes accessible to oxidation into 5hmC. In summary, our data suggest an important role of 5hmC and Tet3 for DNA methylation reprogramming processes in the mammalian zygote. © 2011 Macmillan Publishers Limited. All rights reserved.
Wossidlo M.,Saarland University |
Arand J.,Saarland University |
Sebastiano V.,Max Planck Institute For Molekulare Biomedizin |
Sebastiano V.,Stanford University |
And 5 more authors.
EMBO Journal | Year: 2010
In mammalian zygotes, the 5-methyl-cytosine (5mC) content of paternal chromosomes is rapidly changed by a yet unknown but presumably active enzymatic mechanism. Here, we describe the developmental dynamics and parental asymmetries of DNA methylation in relation to the presence of DNA strand breaks, DNA repair markers and a precise timing of zygotic DNA replication. The analysis shows that distinct pre-replicative (active) and replicative (active and passive) phases of DNA demethylation can be observed. These phases of DNA demethylation are concomitant with the appearance of DNA strand breaks and DNA repair markers such as γH2A.X and PARP-1, respectively. The same correlations are found in cloned embryos obtained after somatic cell nuclear transfer. Together, the data suggest that (1) DNA-methylation reprogramming is more complex and extended as anticipated earlier and (2) the DNA demethylation, particularly the rapid loss of 5mC in paternal DNA, is likely to be linked to DNA repair mechanisms. © 2010 European Molecular Biology Organization.
Lavial F.,Imperial College London |
Bessonnard S.,French Institute of Health and Medical Research |
Ohnishi Y.,Max Planck Institute For Molekulare Biomedizin |
Ohnishi Y.,European Molecular Biology Laboratory |
And 12 more authors.
Genes and Development | Year: 2012
The transcription factors Nanog and Gata6 are critical to specify the epiblast versus primitive endoderm (PrE) lineages. However, little is known about the mechanisms that regulate the protein stability and activity of these factors in the developing embryo. Here we uncover an early developmental function for the Polycomb group member Bmi1 in supporting PrE lineage formation through Gata6 protein stabilization. We show that Bmi1 is enriched in the extraembryonic (endoderm [XEN] and trophectodermal stem [TS]) compartment and repressed by Nanog in pluripotent embryonic stem (ES) cells. In vivo, Bmi1 overlaps with the nascent Gata6 and Nanog protein from the eight-cell stage onward before it preferentially cosegregates with Gata6 in PrE progenitors. Mechanistically, we demonstrate that Bmi1 interacts with Gata6 in a Ring finger-dependent manner to confer protection against Gata6 ubiquitination and proteasomal degradation. A direct role for Bmi1 in cell fate allocation is established by loss-of-function experiments in chimeric embryoid bodies. We thus propose a novel regulatory pathway by which Bmi1 action on Gata6 stability could alter the balance between Gata6 and Nanog protein levels to introduce a bias toward a PrE identity in a cell-autonomous manner. © 2012 by Cold Spring Harbor Laboratory Press.
Antonchick A.P.,Max Planck Institute For Molekulare Physiologie |
Lopez-Tosco S.,Max Planck Institute For Molekulare Physiologie |
Parga J.,Max Planck Institute For Molekulare Biomedizin |
Sievers S.,Max Planck Institute For Molekulare Physiologie |
And 9 more authors.
Chemistry and Biology | Year: 2013
Natural products endowed with neuromodulatory activity and their underlying structural scaffolds may inspire the synthesis of novel neurotrophic compound classes. The spirocyclic secoyohimbane alkaloid rhynchophylline is the major component of the extracts of Uncaria species used in Chinese traditional medicine for treatment of disorders of the central nervous system. Based on the structure of rhynchophylline, a highly enantioselective and efficient organocatalyzed synthesis method was developed that gives access to the tetracyclic secoyohimbane scaffold, embodying a quaternary and three tertiary stereogenic centers in a one-pot multistep reaction sequence. Investigation of a collection of the secoyohimbanes in primary rat hippocampal neurons and embryonal stem cell-derived motor neurons led to discovery of compounds that promote neurite outgrowth and influence the complexity of neuronal network formation. © 2013 Elsevier Ltd.
Schmitz H.,Max Planck Institute For Molekulare Biomedizin |
Stehling M.,Max Planck Institute For Molekulare Biomedizin |
Gentile L.,Max Planck Institute For Molekulare Biomedizin
BioSpektrum | Year: 2014
Flow cytometry is by far the most sophisticated and accurate method for sorting of living stem cells. In general, the target cells need to be labeled for various cell identity markers, which are exposed on the surface of the cells. However, in non-model organisms, we usually lack specific labels for such cell surface markers. Here we describe a method for isolating stem cells from planarians with flow cytometry, based on physiological and morphological properties of these cells. © 2014 Springer-Verlag Berlin Heidelberg. Literatur:.
Hoffmann M.-C.,Ruhr University Bochum |
Muller A.,Ruhr University Bochum |
Fehringer M.,Ruhr University Bochum |
Fehringer M.,Max Planck Institute For Molekulare Biomedizin |
And 3 more authors.
Journal of Bacteriology | Year: 2014
Rhodobacter capsulatus is able to grow with N2 as the sole nitrogen source using either a molybdenum-dependent or a molybdenum-free iron-only nitrogenase whose expression is strictly inhibited by ammonium. Disruption of the fdxD gene, which is located directly upstream of the Mo-nitrogenase genes, nifHDK, abolished diazotrophic growth via Mo-nitrogenase at oxygen concentrations still tolerated by the wild type, thus demonstrating the importance of FdxD under semiaerobic conditions. In contrast, FdxD was not beneficial for diazotrophic growth depending on Fe-nitrogenase. These findings suggest that the 2Fe2S ferredoxin FdxD specifically supports the Mo-nitrogenase system, probably by protecting Mo-nitrogenase against oxygen, as previously shown for its Azotobacter vinelandii counterpart, FeSII. Expression of fdxD occurred under nitrogen-fixing conditions, but not in the presence of ammonium. Expression of fdxD strictly required NifA1 and NifA2, the transcriptional activators of the Mo-nitrogenase genes, but not AnfA, the transcriptional activator of the Fe-nitrogenase genes. Expression of the fdxD and nifH genes, as well as the FdxD and NifH protein levels, increased with increasing molybdate concentrations. Molybdate induction of fdxD was independent of the molybdate-sensing regulators MopA and MopB, which repress anfA transcription at micromolar molybdate concentrations. In this report, we demonstrate the physiological relevance of an fesII-like gene, fdxD, and show that the cellular nitrogen and molybdenum statuses are integrated to control its expression. © 2014, American Society for Microbiology.
Ultramicroscopic visualization of lymph vessels during embryonic development and in the adult mouse [Ultramikroskopische Darstellung von Lymphgefäßen während der Fötalentwicklung und im Erwachsenenstadium der Maus]
Pollmann C.,Max Planck Institute For Molekulare Biomedizin |
Hagerling R.,Max Planck Institute For Molekulare Biomedizin |
Andreas M.,Max Planck Institute For Molekulare Biomedizin |
Kiefer F.,Max Planck Institute For Molekulare Biomedizin
Lymphologie in Forschung und Praxis | Year: 2013
In addition to the circulatory blood vascular system, higher vertebrates rely on a second vascular system - the lymphatic vasculature. Lymphatic vessels are indispensable for tissue homeostasis, uptake of lipids and immune cell circulation. Dysfunction of the lymphatic system may result in immune disorders and edema. During embryogenesis, lymphatic endothelial cells (LECs) differentiate from venous blood endothelium by expression of LEC-specific genes. Subsequently, these cells separate from their venous vessels and give rise to primary lymphatic vessels. Previously, visualization of this process was limited to microscopy of histological tissue sections, precluding comprehensive three-dimensional (3-D) reconstruction at cellular resolution. Using light sheet microscopy, we were able to develop a 3-D ultramicroscopic model of initial lymphangiogenesis in the mouse embryo and show that initial LECs leave the cardinal vein as cords of nonlumenized cells forming a meshwork. Cells in the meshwork subsequently aggregate and give rise to the previously unidentified lymphatic structures, the peripheral longitudinal lymphatic vessel (PLLV) and the primordial thoracic duct (pTD). From these lumenized structures, superficial lymphatic vessels sprout to populate other organ systems, including the skin. As lymphangiogenesis occurs not only during fetal development, but also during adult life under conditions of inflammation, wound healing or tumor formation, intravital visualization of lymphatics and lymphangiogenic processes is a prerequisite for understanding the physiology and pathophysiology of lymphatic vessels. To address this question, we have generated a LEC-specific reporter mouse, which expresses the fluorescent protein mOrange2 under the control of the lymphatic transcription factor Proxl. Using this ProxlmOrange2pA reporter line, optical windows such as the mouse dorsal skinfold chamber and 2-photon microscopy, we are now able to perform intravital studies of lymph flow, lymphatic valve formation, maintenance and function.