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Context-Dependent Functional Divergence of the Notch Ligands DLL1 and DLL4 In Vivo.

Preuße K, Tveriakhina L, Schuster-Gossler K, Gaspar C, Rosa AI, Henrique D, Gossler A, Stauber M - PLoS Genet. (2015)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Institut für Molekularbiologie OE5250, Medizinische Hochschule Hannover, Hannover, Germany.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus

Generation of Dll1Dll4ki mice that express Dll4 instead of Dll1 in the endogenous Dll1 domains.(A) Targeting strategy to insert a Dll4 mini gene into the Dll1 locus. The Dll1 locus contains 11 exons depicted as black boxes (UTRs as white boxes). The targeting construct is comprised of the Dll4 mini gene [Dll4 cDNA from start codon (ATG) in exon 1 to exon 9 (large red box), Dll1 intron 9, Dll4 exon 10 (small red box), Dll1 intron 10 and Dll1 exon 11 that encodes only the terminal valine conserved between Dll1 and Dll4 followed by STOP codon and 3‘UTR], a floxed neor cassette, homology regions for integration between Dll1 start codon and exon 2, and flanking diphtheria toxin genes (DT); insertion of the mini gene is expected to disrupt expression of Dll1. neor is removed by Cre-recombination. The resulting Dll1Dll4ki allele and the Dll1Dll1ki control are shown below (blue boxes, Dll1 mini gene). (B) Heterozygous adult Dll1Dll4ki mice frequently (89%) displayed a kinky tail (arrow in b) but looked otherwise normal. (C) Heterozygous E15.5 Dll1Dll4ki foetuses (c) were indistinguishable from wildtype (wt; a) and homozygous Dll1Dll1ki (b) foetuses while all homozygous Dll1Dll4ki foetuses (d) displayed shortened body axes and large oedemas. (D)Dll1 and Dll4 expression in Dll1Dll4ki and Dll1Dll1ki embryos visualised by whole mount in situ hybridisation of E9.5 embryos of the indicated genotype with a Dll4 ORF, Dll1 ex11 (recognises transcripts from both mini genes) and Dll1 ORF probe confirmed that Dll4ki alleles expressed Dll4 but not Dll1 in Dll1 expression domains (here the PSM, arrowheads). a-c were stained in parallel and colour development was stopped before endogenous Dll4 expression [49] and background became visible. Homozygous Dll1Dll4ki embryos show strong expression in neuroectoderm (white arrow in c; not visible in the weaker staining with Dll1 ex11 probe in f). (E) Northern blot analysis of homozygous Dll1Dll4ki and Dll1Dll1ki E11.5 embryos, 2 μg polyA(+)-RNA loaded per lane, hybridised with 3‘UTR (Dll1 ex11) and β-actin probes; quantification of transgene signals relative to actin is shown at the bottom and indicates similar expression levels. (F) Visualisation of DLL1 and DLL4 expressed in the PSM of homozygous Dll1Dll4ki (a-f) and Dll1Dll1ki E9.5 embryos (g-l) using specific anti-DLL1 and anti-DLL4 antibodies. Co-staining with anti-panCadherin antibodies, which mark the plasma membrane, confirms that transgenic DLL4 and DLL1 predominantly localise to the cell surface (c,l). The lack of DLL1 signal in Dll1Dll4ki (d) and of DLL4 signal in Dll1Dll1ki PSMs (g) confirm the specificity of stainings. Both in anti-DLL4 and anti-DLL1 antibody stainings of PSMs, we observed spots of high signal intensity that may result from accumulation of ligands at these sites and that had also been observed in wildtype PSMs stained with anti-DLL1 antibodies [21]. Scale bars, 10 μm; insets show magnifications of the dotted boxes in c,l.
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pgen.1005328.g002: Generation of Dll1Dll4ki mice that express Dll4 instead of Dll1 in the endogenous Dll1 domains.(A) Targeting strategy to insert a Dll4 mini gene into the Dll1 locus. The Dll1 locus contains 11 exons depicted as black boxes (UTRs as white boxes). The targeting construct is comprised of the Dll4 mini gene [Dll4 cDNA from start codon (ATG) in exon 1 to exon 9 (large red box), Dll1 intron 9, Dll4 exon 10 (small red box), Dll1 intron 10 and Dll1 exon 11 that encodes only the terminal valine conserved between Dll1 and Dll4 followed by STOP codon and 3‘UTR], a floxed neor cassette, homology regions for integration between Dll1 start codon and exon 2, and flanking diphtheria toxin genes (DT); insertion of the mini gene is expected to disrupt expression of Dll1. neor is removed by Cre-recombination. The resulting Dll1Dll4ki allele and the Dll1Dll1ki control are shown below (blue boxes, Dll1 mini gene). (B) Heterozygous adult Dll1Dll4ki mice frequently (89%) displayed a kinky tail (arrow in b) but looked otherwise normal. (C) Heterozygous E15.5 Dll1Dll4ki foetuses (c) were indistinguishable from wildtype (wt; a) and homozygous Dll1Dll1ki (b) foetuses while all homozygous Dll1Dll4ki foetuses (d) displayed shortened body axes and large oedemas. (D)Dll1 and Dll4 expression in Dll1Dll4ki and Dll1Dll1ki embryos visualised by whole mount in situ hybridisation of E9.5 embryos of the indicated genotype with a Dll4 ORF, Dll1 ex11 (recognises transcripts from both mini genes) and Dll1 ORF probe confirmed that Dll4ki alleles expressed Dll4 but not Dll1 in Dll1 expression domains (here the PSM, arrowheads). a-c were stained in parallel and colour development was stopped before endogenous Dll4 expression [49] and background became visible. Homozygous Dll1Dll4ki embryos show strong expression in neuroectoderm (white arrow in c; not visible in the weaker staining with Dll1 ex11 probe in f). (E) Northern blot analysis of homozygous Dll1Dll4ki and Dll1Dll1ki E11.5 embryos, 2 μg polyA(+)-RNA loaded per lane, hybridised with 3‘UTR (Dll1 ex11) and β-actin probes; quantification of transgene signals relative to actin is shown at the bottom and indicates similar expression levels. (F) Visualisation of DLL1 and DLL4 expressed in the PSM of homozygous Dll1Dll4ki (a-f) and Dll1Dll1ki E9.5 embryos (g-l) using specific anti-DLL1 and anti-DLL4 antibodies. Co-staining with anti-panCadherin antibodies, which mark the plasma membrane, confirms that transgenic DLL4 and DLL1 predominantly localise to the cell surface (c,l). The lack of DLL1 signal in Dll1Dll4ki (d) and of DLL4 signal in Dll1Dll1ki PSMs (g) confirm the specificity of stainings. Both in anti-DLL4 and anti-DLL1 antibody stainings of PSMs, we observed spots of high signal intensity that may result from accumulation of ligands at these sites and that had also been observed in wildtype PSMs stained with anti-DLL1 antibodies [21]. Scale bars, 10 μm; insets show magnifications of the dotted boxes in c,l.

Mentions: To further investigate to which degree DLL4 can compensate for the loss of DLL1 during somite patterning and in other developmental contexts, we generated mice that express DLL4 from the Dll1 locus instead of endogenous DLL1. To replace endogenous Dll1 with Dll4, we applied a knock-in strategy inserting a Dll4 mini gene into the first and second exons of Dll1 (Fig 2A). Production of DLL4 protein of the correct size from the Dll4 mini gene was confirmed by Western blot analysis of lysates of CHO cells transiently expressing the Dll4 mini gene (S2 Fig). We generated mice carrying the Dll4 mini gene in the Dll1 locus, referred to as Dll1Dll4ki. As a control, we used the analogous knock-in of a Dll1 mini gene into the Dll1 locus (Fig 2A bottom; Dll1tm2Gos, here referred to as Dll1Dll1ki), which was identical to the Dll4 mini gene with regard to its exon/intron structure, intron sequences and the 5' and 3' UTRs but encoded DLL1. Homozygous Dll1Dll1ki mice were viable and fertile and appeared phenotypically normal indicating that the Dll1 mini gene can functionally substitute the endogenous Dll1 gene [37].


Context-Dependent Functional Divergence of the Notch Ligands DLL1 and DLL4 In Vivo.

Preuße K, Tveriakhina L, Schuster-Gossler K, Gaspar C, Rosa AI, Henrique D, Gossler A, Stauber M - PLoS Genet. (2015)

Generation of Dll1Dll4ki mice that express Dll4 instead of Dll1 in the endogenous Dll1 domains.(A) Targeting strategy to insert a Dll4 mini gene into the Dll1 locus. The Dll1 locus contains 11 exons depicted as black boxes (UTRs as white boxes). The targeting construct is comprised of the Dll4 mini gene [Dll4 cDNA from start codon (ATG) in exon 1 to exon 9 (large red box), Dll1 intron 9, Dll4 exon 10 (small red box), Dll1 intron 10 and Dll1 exon 11 that encodes only the terminal valine conserved between Dll1 and Dll4 followed by STOP codon and 3‘UTR], a floxed neor cassette, homology regions for integration between Dll1 start codon and exon 2, and flanking diphtheria toxin genes (DT); insertion of the mini gene is expected to disrupt expression of Dll1. neor is removed by Cre-recombination. The resulting Dll1Dll4ki allele and the Dll1Dll1ki control are shown below (blue boxes, Dll1 mini gene). (B) Heterozygous adult Dll1Dll4ki mice frequently (89%) displayed a kinky tail (arrow in b) but looked otherwise normal. (C) Heterozygous E15.5 Dll1Dll4ki foetuses (c) were indistinguishable from wildtype (wt; a) and homozygous Dll1Dll1ki (b) foetuses while all homozygous Dll1Dll4ki foetuses (d) displayed shortened body axes and large oedemas. (D)Dll1 and Dll4 expression in Dll1Dll4ki and Dll1Dll1ki embryos visualised by whole mount in situ hybridisation of E9.5 embryos of the indicated genotype with a Dll4 ORF, Dll1 ex11 (recognises transcripts from both mini genes) and Dll1 ORF probe confirmed that Dll4ki alleles expressed Dll4 but not Dll1 in Dll1 expression domains (here the PSM, arrowheads). a-c were stained in parallel and colour development was stopped before endogenous Dll4 expression [49] and background became visible. Homozygous Dll1Dll4ki embryos show strong expression in neuroectoderm (white arrow in c; not visible in the weaker staining with Dll1 ex11 probe in f). (E) Northern blot analysis of homozygous Dll1Dll4ki and Dll1Dll1ki E11.5 embryos, 2 μg polyA(+)-RNA loaded per lane, hybridised with 3‘UTR (Dll1 ex11) and β-actin probes; quantification of transgene signals relative to actin is shown at the bottom and indicates similar expression levels. (F) Visualisation of DLL1 and DLL4 expressed in the PSM of homozygous Dll1Dll4ki (a-f) and Dll1Dll1ki E9.5 embryos (g-l) using specific anti-DLL1 and anti-DLL4 antibodies. Co-staining with anti-panCadherin antibodies, which mark the plasma membrane, confirms that transgenic DLL4 and DLL1 predominantly localise to the cell surface (c,l). The lack of DLL1 signal in Dll1Dll4ki (d) and of DLL4 signal in Dll1Dll1ki PSMs (g) confirm the specificity of stainings. Both in anti-DLL4 and anti-DLL1 antibody stainings of PSMs, we observed spots of high signal intensity that may result from accumulation of ligands at these sites and that had also been observed in wildtype PSMs stained with anti-DLL1 antibodies [21]. Scale bars, 10 μm; insets show magnifications of the dotted boxes in c,l.
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pgen.1005328.g002: Generation of Dll1Dll4ki mice that express Dll4 instead of Dll1 in the endogenous Dll1 domains.(A) Targeting strategy to insert a Dll4 mini gene into the Dll1 locus. The Dll1 locus contains 11 exons depicted as black boxes (UTRs as white boxes). The targeting construct is comprised of the Dll4 mini gene [Dll4 cDNA from start codon (ATG) in exon 1 to exon 9 (large red box), Dll1 intron 9, Dll4 exon 10 (small red box), Dll1 intron 10 and Dll1 exon 11 that encodes only the terminal valine conserved between Dll1 and Dll4 followed by STOP codon and 3‘UTR], a floxed neor cassette, homology regions for integration between Dll1 start codon and exon 2, and flanking diphtheria toxin genes (DT); insertion of the mini gene is expected to disrupt expression of Dll1. neor is removed by Cre-recombination. The resulting Dll1Dll4ki allele and the Dll1Dll1ki control are shown below (blue boxes, Dll1 mini gene). (B) Heterozygous adult Dll1Dll4ki mice frequently (89%) displayed a kinky tail (arrow in b) but looked otherwise normal. (C) Heterozygous E15.5 Dll1Dll4ki foetuses (c) were indistinguishable from wildtype (wt; a) and homozygous Dll1Dll1ki (b) foetuses while all homozygous Dll1Dll4ki foetuses (d) displayed shortened body axes and large oedemas. (D)Dll1 and Dll4 expression in Dll1Dll4ki and Dll1Dll1ki embryos visualised by whole mount in situ hybridisation of E9.5 embryos of the indicated genotype with a Dll4 ORF, Dll1 ex11 (recognises transcripts from both mini genes) and Dll1 ORF probe confirmed that Dll4ki alleles expressed Dll4 but not Dll1 in Dll1 expression domains (here the PSM, arrowheads). a-c were stained in parallel and colour development was stopped before endogenous Dll4 expression [49] and background became visible. Homozygous Dll1Dll4ki embryos show strong expression in neuroectoderm (white arrow in c; not visible in the weaker staining with Dll1 ex11 probe in f). (E) Northern blot analysis of homozygous Dll1Dll4ki and Dll1Dll1ki E11.5 embryos, 2 μg polyA(+)-RNA loaded per lane, hybridised with 3‘UTR (Dll1 ex11) and β-actin probes; quantification of transgene signals relative to actin is shown at the bottom and indicates similar expression levels. (F) Visualisation of DLL1 and DLL4 expressed in the PSM of homozygous Dll1Dll4ki (a-f) and Dll1Dll1ki E9.5 embryos (g-l) using specific anti-DLL1 and anti-DLL4 antibodies. Co-staining with anti-panCadherin antibodies, which mark the plasma membrane, confirms that transgenic DLL4 and DLL1 predominantly localise to the cell surface (c,l). The lack of DLL1 signal in Dll1Dll4ki (d) and of DLL4 signal in Dll1Dll1ki PSMs (g) confirm the specificity of stainings. Both in anti-DLL4 and anti-DLL1 antibody stainings of PSMs, we observed spots of high signal intensity that may result from accumulation of ligands at these sites and that had also been observed in wildtype PSMs stained with anti-DLL1 antibodies [21]. Scale bars, 10 μm; insets show magnifications of the dotted boxes in c,l.
Mentions: To further investigate to which degree DLL4 can compensate for the loss of DLL1 during somite patterning and in other developmental contexts, we generated mice that express DLL4 from the Dll1 locus instead of endogenous DLL1. To replace endogenous Dll1 with Dll4, we applied a knock-in strategy inserting a Dll4 mini gene into the first and second exons of Dll1 (Fig 2A). Production of DLL4 protein of the correct size from the Dll4 mini gene was confirmed by Western blot analysis of lysates of CHO cells transiently expressing the Dll4 mini gene (S2 Fig). We generated mice carrying the Dll4 mini gene in the Dll1 locus, referred to as Dll1Dll4ki. As a control, we used the analogous knock-in of a Dll1 mini gene into the Dll1 locus (Fig 2A bottom; Dll1tm2Gos, here referred to as Dll1Dll1ki), which was identical to the Dll4 mini gene with regard to its exon/intron structure, intron sequences and the 5' and 3' UTRs but encoded DLL1. Homozygous Dll1Dll1ki mice were viable and fertile and appeared phenotypically normal indicating that the Dll1 mini gene can functionally substitute the endogenous Dll1 gene [37].

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Institut für Molekularbiologie OE5250, Medizinische Hochschule Hannover, Hannover, Germany.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus