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Preusse K.,Institute For Molekularbiologie Oe5250 | Tveriakhina L.,Institute For Molekularbiologie Oe5250 | Schuster-Gossler K.,Institute For Molekularbiologie Oe5250 | Gaspar C.,University of Lisbon | And 4 more authors.
PLoS Genetics | Year: 2015

Notch signalling is a fundamental pathway that shapes the developing embryo and sustains adult tissues by direct communication between ligand and receptor molecules on adjacent cells. Among the ligands are two Delta paralogues, DLL1 and DLL4, that are conserved in mammals and share a similar structure and sequence. They activate the Notch receptor partly in overlapping expression domains where they fulfil redundant functions in some processes (e.g. maintenance of the crypt cell progenitor pool). In other processes, however, they appear to act differently (e.g. maintenance of foetal arterial identity) raising the questions of how similar DLL1 and DLL4 really are and which mechanism causes the apparent context-dependent divergence. By analysing mice that conditionally overexpress DLL1 or DLL4 from the same genomic locus (Hprt) and mice that express DLL4 instead of DLL1 from the endogenous Dll1 locus (Dll1Dll4ki), we found functional differences that are tissue-specific: while DLL1 and DLL4 act redundantly during the maintenance of retinal progenitors, their function varies in the presomitic mesoderm (PSM) where somites form in a Notch-dependent process. In the anterior PSM, every cell expresses both Notch receptors and ligands, and DLL1 is the only activator of Notch while DLL4 is not endogenously expressed. Transgenic DLL4 cannot replace DLL1 during somitogenesis and in heterozygous Dll1Dll4ki/+ mice, the Dll1Dll4ki allele causes a dominant segmentation phenotype. Testing several aspects of the complex Notch signalling system in vitro, we found that both ligands have a similar trans-activation potential but that only DLL4 is an efficient cis-inhibitor of Notch signalling, causing a reduced net activation of Notch. These differential cis-inhibitory properties are likely to contribute to the functional divergence of DLL1 and DLL4. © 2015 Preuße et al.


PubMed | Institute For Molekularbiologie Oe5250 and University of Lisbon
Type: Journal Article | Journal: PLoS genetics | Year: 2015

Notch signalling is a fundamental pathway that shapes the developing embryo and sustains adult tissues by direct communication between ligand and receptor molecules on adjacent cells. Among the ligands are two Delta paralogues, DLL1 and DLL4, that are conserved in mammals and share a similar structure and sequence. They activate the Notch receptor partly in overlapping expression domains where they fulfil redundant functions in some processes (e.g. maintenance of the crypt cell progenitor pool). In other processes, however, they appear to act differently (e.g. maintenance of foetal arterial identity) raising the questions of how similar DLL1 and DLL4 really are and which mechanism causes the apparent context-dependent divergence. By analysing mice that conditionally overexpress DLL1 or DLL4 from the same genomic locus (Hprt) and mice that express DLL4 instead of DLL1 from the endogenous Dll1 locus (Dll1Dll4ki), we found functional differences that are tissue-specific: while DLL1 and DLL4 act redundantly during the maintenance of retinal progenitors, their function varies in the presomitic mesoderm (PSM) where somites form in a Notch-dependent process. In the anterior PSM, every cell expresses both Notch receptors and ligands, and DLL1 is the only activator of Notch while DLL4 is not endogenously expressed. Transgenic DLL4 cannot replace DLL1 during somitogenesis and in heterozygous Dll1Dll4ki/+ mice, the Dll1Dll4ki allele causes a dominant segmentation phenotype. Testing several aspects of the complex Notch signalling system in vitro, we found that both ligands have a similar trans-activation potential but that only DLL4 is an efficient cis-inhibitor of Notch signalling, causing a reduced net activation of Notch. These differential cis-inhibitory properties are likely to contribute to the functional divergence of DLL1 and DLL4.


Serth K.,Institute For Molekularbiologie Oe5250 | Schuster-Gossler K.,Institute For Molekularbiologie Oe5250 | Kremmer E.,Helmholtz Center for Environmental Research | Hansen B.,Institute For Molekularbiologie Oe5250 | And 3 more authors.
PLoS ONE | Year: 2015

Delta-like 3 (DLL3) is a member of the DSL family of Notch ligands in amniotes. In contrast to DLL1 and DLL4, the other Delta-like proteins in the mouse, DLL3 does not bind in trans to Notch and does not activate the receptor, but shows cis-interaction and cis-inhibitory properties on Notch signaling in vitro. Loss of the DSL protein DLL3 in the mouse results in severe somite patterning defects, which are virtually indistinguishable from the defects in mice that lack lunatic fringe (LFNG), a glycosyltransferase involved in modifying Notch signaling. Like LFNG, DLL3 is located within the trans-Golgi, however, its biochemical function is still unclear. Here, we show that i) both proteins interact, ii) epidermal growth factor like repeats 2 and 5 of DLL3 are O-fucosylated at consensus sites for POFUT1, and iii) further modified by FNG proteins in vitro. Embryos double homozygous for null mutations in Dll3 and Lfng are phenotypically indistinguishable from the single mutants supporting a potential common function. Mutation of the O-fucosylation sites in DLL3 does not disrupt the interaction of DLL3 with LFNG or full length Notch1or DLL1, and O-fucosylation-deficient DLL3 can still inhibit Notch in cis in vitro. However, in contrast to wild type DLL3, O-fucosylation-deficient DLL3 cannot compensate for the loss of endogenous DLL3 during somitogenesis in the embryo. Together our results suggest that the cis-inhibitory activity of DLL3 observed in cultured cells might not fully reflect its assumed essential physiological property, suggest that DLL3 and LFNG act together, and strongly supports that modification of DLL3 by O-linked fucose is essential for its function during somitogenesis. © 2015 Serth et al.


Drusenheimer N.,Heinrich Heine University Düsseldorf | Migdal B.,Heinrich Heine University Düsseldorf | Jackel S.,German Center for Neurodegenerative Diseases | Tveriakhina L.,Institute For Molekularbiologie Oe5250 | And 5 more authors.
PLoS Genetics | Year: 2015

CC2D1A and CC2D1B belong to the evolutionary conserved Lgd protein family with members in all multi-cellular animals. Several functions such as centrosomal cleavage, involvement in signalling pathways, immune response and synapse maturation have been described for CC2D1A. Moreover, the Drosophila melanogaster ortholog Lgd was shown to be involved in the endosomal trafficking of the Notch receptor and other transmembrane receptors and physically interacts with the ESCRT-III component Shrub/CHMP4. To determine if this function is conserved in mammals we generated and characterized Cc2d1a and Cc2d1b conditional knockout mice. While Cc2d1b deficient mice displayed no obvious phenotype, we found that Cc2d1a deficient mice as well as conditional mutants that lack CC2D1A only in the nervous system die shortly after birth due to respiratory distress. This finding confirms the suspicion that the breathing defect is caused by the central nervous system. However, an involvement in centrosomal function could not be confirmed in Cc2d1a deficient MEF cells. To analyse an influence on Notch signalling, we generated intestine specific Cc2d1a mutant mice. These mice did not display any alterations in goblet cell number, proliferating cell number or expression of the Notch reporter Hes1-emGFP, suggesting that CC2D1A is not required for Notch signalling. However, our EM analysis revealed that the average size of endosomes of Cc2d1a mutant cells, but not Cc2d1b mutant cells, is increased, indicating a defect in endosomal morphogenesis. We could show that CC2D1A and its interaction partner CHMP4B are localised on endosomes in MEF cells, when the activity of the endosomal protein VPS4 is reduced. This indicates that CC2D1A cycles between the cytosol and the endosomal membrane. Additionally, in rescue experiments in D. melanogaster, CC2D1A and CC2D1B were able to functionally replace Lgd. Altogether our data suggest a functional conservation of the Lgd protein family in the ESCRT-III mediated process in metazoans. © 2015 Drusenheimer et al.


PubMed | Institute For Molekularbiologie Oe5250, Heinrich Heine University Düsseldorf and German Center for Neurodegenerative Diseases
Type: Journal Article | Journal: PLoS genetics | Year: 2016

CC2D1A and CC2D1B belong to the evolutionary conserved Lgd protein family with members in all multi-cellular animals. Several functions such as centrosomal cleavage, involvement in signalling pathways, immune response and synapse maturation have been described for CC2D1A. Moreover, the Drosophila melanogaster ortholog Lgd was shown to be involved in the endosomal trafficking of the Notch receptor and other transmembrane receptors and physically interacts with the ESCRT-III component Shrub/CHMP4. To determine if this function is conserved in mammals we generated and characterized Cc2d1a and Cc2d1b conditional knockout mice. While Cc2d1b deficient mice displayed no obvious phenotype, we found that Cc2d1a deficient mice as well as conditional mutants that lack CC2D1A only in the nervous system die shortly after birth due to respiratory distress. This finding confirms the suspicion that the breathing defect is caused by the central nervous system. However, an involvement in centrosomal function could not be confirmed in Cc2d1a deficient MEF cells. To analyse an influence on Notch signalling, we generated intestine specific Cc2d1a mutant mice. These mice did not display any alterations in goblet cell number, proliferating cell number or expression of the Notch reporter Hes1-emGFP, suggesting that CC2D1A is not required for Notch signalling. However, our EM analysis revealed that the average size of endosomes of Cc2d1a mutant cells, but not Cc2d1b mutant cells, is increased, indicating a defect in endosomal morphogenesis. We could show that CC2D1A and its interaction partner CHMP4B are localised on endosomes in MEF cells, when the activity of the endosomal protein VPS4 is reduced. This indicates that CC2D1A cycles between the cytosol and the endosomal membrane. Additionally, in rescue experiments in D. melanogaster, CC2D1A and CC2D1B were able to functionally replace Lgd. Altogether our data suggest a functional conservation of the Lgd protein family in the ESCRT-III mediated process in metazoans.


PubMed | Institute For Molekularbiologie Oe5250 and Helmholtz Center for Environmental Research
Type: Journal Article | Journal: PloS one | Year: 2015

Delta-like 3 (DLL3) is a member of the DSL family of Notch ligands in amniotes. In contrast to DLL1 and DLL4, the other Delta-like proteins in the mouse, DLL3 does not bind in trans to Notch and does not activate the receptor, but shows cis-interaction and cis-inhibitory properties on Notch signaling in vitro. Loss of the DSL protein DLL3 in the mouse results in severe somite patterning defects, which are virtually indistinguishable from the defects in mice that lack lunatic fringe (LFNG), a glycosyltransferase involved in modifying Notch signaling. Like LFNG, DLL3 is located within the trans-Golgi, however, its biochemical function is still unclear. Here, we show that i) both proteins interact, ii) epidermal growth factor like repeats 2 and 5 of DLL3 are O-fucosylated at consensus sites for POFUT1, and iii) further modified by FNG proteins in vitro. Embryos double homozygous for null mutations in Dll3 and Lfng are phenotypically indistinguishable from the single mutants supporting a potential common function. Mutation of the O-fucosylation sites in DLL3 does not disrupt the interaction of DLL3 with LFNG or full length Notch1or DLL1, and O-fucosylation-deficient DLL3 can still inhibit Notch in cis in vitro. However, in contrast to wild type DLL3, O-fucosylation-deficient DLL3 cannot compensate for the loss of endogenous DLL3 during somitogenesis in the embryo. Together our results suggest that the cis-inhibitory activity of DLL3 observed in cultured cells might not fully reflect its assumed essential physiological property, suggest that DLL3 and LFNG act together, and strongly supports that modification of DLL3 by O-linked fucose is essential for its function during somitogenesis.


Serth K.,Institute For Molekularbiologie Oe5250 | Beckers A.,Institute For Molekularbiologie Oe5250 | Schuster-Gossler K.,Institute For Molekularbiologie Oe5250 | Pavlova M.N.,Institute For Molekularbiologie Oe5250 | And 6 more authors.
BMC Genetics | Year: 2015

Background: Etl4lacZ (Enhancer trap locus 4) and SktGt (Sickle tail) are lacZ reporter gene integrations into the same locus on mouse chromosome 2 targeting a gene that is expressed in the notochord of early embryos and in multiple epithelia during later development. Both insertions caused recessive mutations that resulted exclusively in mild defects in the caudal vertebral column. Since notochord-derived signals are essential for formation of the vertebral column the phenotypes suggested that the lacZ insertions interfered with some notochord-dependent aspect of vertebral development. As both insertions occurred in introns it was unclear whether they represent hypomorphic alleles or abolish gene function. Here, we have generated a definitive null allele of the Skt/Etl4 gene and analysed homozygous mutants. Results: We have introduced loxP sites into three positions of the gene based on additional upstream exons that we identified, and deleted approximately 870 kb of the locus by a combination of inter- and intra-chromosomal Cre-mediated recombinations in the female germ line of mice. This deletion removes about 90 % of the coding region and results in the loss of the SKT/ETL4 protein. Similar to the Etl4lacZ and SktGt alleles our deletion mutants are viable and fertile and show only mild defects in caudal vertebrae due to abnormal intervertebral disc development, although with higher penetrance. No other tissue with Skt/Etl4 expression that we analysed showed obvious defects. Conclusion: The complete loss of Skt/Etl4 function affects only development of caudal notochord derivatives and is compensated for in its other expression domains. © 2015 Serth et al.


Redeker C.,Institute For Molekularbiologie Oe5250 | Schuster-Gossler K.,Institute For Molekularbiologie Oe5250 | Kremmer E.,Helmholtz Center Munich | Gossler A.,Institute For Molekularbiologie Oe5250
PLoS ONE | Year: 2013

The Notch signaling pathway mediates the direct communication between adjacent cells and regulates multiple developmental processes. Interaction of the Notch receptor with its ligands induces the liberation of the intracellular portion of Notch (NICD) referred to as regulated intramembraneous proteolysis (RIP). NICD translocates to the nucleus, and by complexing with the DNA binding protein RBPjκ and other cofactors activates transcription of bHLH genes. RIP-like processing of various mammalian Notch ligands (DLL1, JAG1 and JAG2) and the translocation of their intracellular domains (ICDs) to the nucleus has also been observed. These observations together with effects of over-expressed ligand ICDs in cultured cells on cell proliferation, differentiation, and Notch activity and target gene expression have led to the idea that the intracellular domains of Notch ligands have signaling functions. To test this hypothesis in vivo we have generated ES cells and transgenic mice that constitutively express various versions of the intracellular domain of mouse DLL1. In contrast to other cell lines, expression of DICDs in ES cells did not block proliferation or stimulate neuronal differentiation. Embryos with ubiquitous DICD expression developed to term without any apparent phenotype and grew up to viable and fertile adults. Early Notch-dependent processes or expression of selected Notch target genes were unaltered in transgenic embryos. In addition, we show that mouse DICD enters the nucleus inefficiently. Collectively, our results argue against a signaling activity of the intracellular domain of DLL1 in mouse embryos in vivo. © 2013 Redeker et al.

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