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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

DLL1 and DLL4 trans-activate Notch with similar efficiency, but only DLL4 is an effective cis-inhibitor.(A) Flag-tagged Dll1 and Dll4 ORFs were inserted into a randomly integrated attP site in CHOattP cells mediated by ΦC31 site-directed recombination (upper part). Resulting cells were used in Notch-activation assays in combination with HeLa-N1 cells as schematically shown below (DLL1 depicted as blue bar; DLL4, red; NOTCH1, grey; HeLa-N1 cells are encircled in green). (B) Quantification of DLL1-Flag and DLL4-Flag in two independent CHOattP-DLL1 (B5, C6) and CHOattP-DLL4 (B5, D3) cell lines by Western blot analysis of cell lysates with anti-Flag and anti-β-actin (for normalisation) antibodies showed similar protein levels. (C) Surface biotinylation assays demonstrated equal surface representation of DLL1 and DLL4 on CHOattP cells. (D) Notch trans-activation assays by co-culture of HeLa-N1 cells containing an RBP-Jκ:Luciferase reporter with CHOattP-DLL1 or CHOattP-DLL4 cells. All DLL1 and DLL4 clones activated Notch similarly, DLL4 being a slightly more efficient activator (compare with similar experiment in S6A and S6G Fig). (E) Notch trans-activation and cis-inhibition assays by culturing HeLa-N1 cells untransfected or transiently transfected with Dll1 or Dll4 expression constructs with or without CHOattP or CHOattP-DLL1 cells as indicated (a-c). Co-culture conditions a, b and c correspond to Luciferase measurements a’, b’ and c’, respectively. Results show cis-inhibition by DLL4 but not DLL1; for details see main text. (F)trans-Activation assays (a) without and (b) with NOTCH1 receptor expression in the signal sending CHO cell to test if NOTCH1 cis-inhibits the ligand activity of DLL1 or DLL4. No cis-inhibitory effect on either ligand was observed (columns a‘ and b‘ correspond to assay conditions a and b, respectively). (G)trans-Activation and cis-inhibition assays using chimeric DLL1-DLL4 proteins (G top; depicted as red and blue striped bars in a-c). HeLa-N1 cells were transiently transfected with no or DLL4-DLL1ECD or DLL1-DLL4ECD expression constructs and cultured as indicated (a-c). Under all three conditions, a strong cis-inhibitory activity was detected only for DLL1-DLL4ECD (columns a‘, b’ and c‘ correspond to schemas a, b and c, respectively). Error bars represent SEM; ns, not significant; *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001.
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pgen.1005328.g005: DLL1 and DLL4 trans-activate Notch with similar efficiency, but only DLL4 is an effective cis-inhibitor.(A) Flag-tagged Dll1 and Dll4 ORFs were inserted into a randomly integrated attP site in CHOattP cells mediated by ΦC31 site-directed recombination (upper part). Resulting cells were used in Notch-activation assays in combination with HeLa-N1 cells as schematically shown below (DLL1 depicted as blue bar; DLL4, red; NOTCH1, grey; HeLa-N1 cells are encircled in green). (B) Quantification of DLL1-Flag and DLL4-Flag in two independent CHOattP-DLL1 (B5, C6) and CHOattP-DLL4 (B5, D3) cell lines by Western blot analysis of cell lysates with anti-Flag and anti-β-actin (for normalisation) antibodies showed similar protein levels. (C) Surface biotinylation assays demonstrated equal surface representation of DLL1 and DLL4 on CHOattP cells. (D) Notch trans-activation assays by co-culture of HeLa-N1 cells containing an RBP-Jκ:Luciferase reporter with CHOattP-DLL1 or CHOattP-DLL4 cells. All DLL1 and DLL4 clones activated Notch similarly, DLL4 being a slightly more efficient activator (compare with similar experiment in S6A and S6G Fig). (E) Notch trans-activation and cis-inhibition assays by culturing HeLa-N1 cells untransfected or transiently transfected with Dll1 or Dll4 expression constructs with or without CHOattP or CHOattP-DLL1 cells as indicated (a-c). Co-culture conditions a, b and c correspond to Luciferase measurements a’, b’ and c’, respectively. Results show cis-inhibition by DLL4 but not DLL1; for details see main text. (F)trans-Activation assays (a) without and (b) with NOTCH1 receptor expression in the signal sending CHO cell to test if NOTCH1 cis-inhibits the ligand activity of DLL1 or DLL4. No cis-inhibitory effect on either ligand was observed (columns a‘ and b‘ correspond to assay conditions a and b, respectively). (G)trans-Activation and cis-inhibition assays using chimeric DLL1-DLL4 proteins (G top; depicted as red and blue striped bars in a-c). HeLa-N1 cells were transiently transfected with no or DLL4-DLL1ECD or DLL1-DLL4ECD expression constructs and cultured as indicated (a-c). Under all three conditions, a strong cis-inhibitory activity was detected only for DLL1-DLL4ECD (columns a‘, b’ and c‘ correspond to schemas a, b and c, respectively). Error bars represent SEM; ns, not significant; *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001.

Mentions: To investigate the functional difference between DLL1 and DLL4 in vitro, we performed co-culture experiments by mixing cells expressing NOTCH1 receptor or DLL ligands and measured Notch activation with a reporter in the receptor-expressing cells. Specifically, we used HeLa cells that express both the NOTCH1 receptor (stable HeLa-N1 cells; [10]) and a transient Notch activity reporter based on an RBP-Jk promoter-driven Luciferase [56] with CHO cells stably expressing Flag-tagged DLL1 or DLL4 ligands. To ensure comparability of results, we integrated single copies of Dll1 or Dll4 ORFs under the control of the CMV promoter into the identical genomic locus of CHO cells by adopting a site-directed attP/attB recombination system (Fig 5A top; S4 Fig; [57]). We established CHO cells with a pre-inserted, randomly integrated single attP site (termed CHOattP; uniqueness of this attP site was confirmed by Southern blot analysis; S4A and S4B Fig) and recombined Dll1 or Dll4 ORFs into this site (cell lines termed CHOattP-DLL1 and CHOattP-DLL4; Fig 5A bottom left). Consistent with the expression from the same genomic locus, independent CHOattP-DLL1 (B5, C6) and CHOattP-DLL4 (B5, D3) clones expressed DLL1 and DLL4 protein at similar levels (Fig 5B; n = 4 lysates of each clone; S5A Fig, S3 Table, S5B Fig, S4 Table) and cell surface representation of DLL1 and DLL4 was similar in all lines (~40%; Fig 5C; n≥3 biotinylation assays; S5C and S5D Fig, S5 Table). Likewise, half-lives of DLL1 and DLL4 proteins were similar, DLL4 being slightly more stable (S5E and S5F Fig).


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)

DLL1 and DLL4 trans-activate Notch with similar efficiency, but only DLL4 is an effective cis-inhibitor.(A) Flag-tagged Dll1 and Dll4 ORFs were inserted into a randomly integrated attP site in CHOattP cells mediated by ΦC31 site-directed recombination (upper part). Resulting cells were used in Notch-activation assays in combination with HeLa-N1 cells as schematically shown below (DLL1 depicted as blue bar; DLL4, red; NOTCH1, grey; HeLa-N1 cells are encircled in green). (B) Quantification of DLL1-Flag and DLL4-Flag in two independent CHOattP-DLL1 (B5, C6) and CHOattP-DLL4 (B5, D3) cell lines by Western blot analysis of cell lysates with anti-Flag and anti-β-actin (for normalisation) antibodies showed similar protein levels. (C) Surface biotinylation assays demonstrated equal surface representation of DLL1 and DLL4 on CHOattP cells. (D) Notch trans-activation assays by co-culture of HeLa-N1 cells containing an RBP-Jκ:Luciferase reporter with CHOattP-DLL1 or CHOattP-DLL4 cells. All DLL1 and DLL4 clones activated Notch similarly, DLL4 being a slightly more efficient activator (compare with similar experiment in S6A and S6G Fig). (E) Notch trans-activation and cis-inhibition assays by culturing HeLa-N1 cells untransfected or transiently transfected with Dll1 or Dll4 expression constructs with or without CHOattP or CHOattP-DLL1 cells as indicated (a-c). Co-culture conditions a, b and c correspond to Luciferase measurements a’, b’ and c’, respectively. Results show cis-inhibition by DLL4 but not DLL1; for details see main text. (F)trans-Activation assays (a) without and (b) with NOTCH1 receptor expression in the signal sending CHO cell to test if NOTCH1 cis-inhibits the ligand activity of DLL1 or DLL4. No cis-inhibitory effect on either ligand was observed (columns a‘ and b‘ correspond to assay conditions a and b, respectively). (G)trans-Activation and cis-inhibition assays using chimeric DLL1-DLL4 proteins (G top; depicted as red and blue striped bars in a-c). HeLa-N1 cells were transiently transfected with no or DLL4-DLL1ECD or DLL1-DLL4ECD expression constructs and cultured as indicated (a-c). Under all three conditions, a strong cis-inhibitory activity was detected only for DLL1-DLL4ECD (columns a‘, b’ and c‘ correspond to schemas a, b and c, respectively). Error bars represent SEM; ns, not significant; *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001.
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pgen.1005328.g005: DLL1 and DLL4 trans-activate Notch with similar efficiency, but only DLL4 is an effective cis-inhibitor.(A) Flag-tagged Dll1 and Dll4 ORFs were inserted into a randomly integrated attP site in CHOattP cells mediated by ΦC31 site-directed recombination (upper part). Resulting cells were used in Notch-activation assays in combination with HeLa-N1 cells as schematically shown below (DLL1 depicted as blue bar; DLL4, red; NOTCH1, grey; HeLa-N1 cells are encircled in green). (B) Quantification of DLL1-Flag and DLL4-Flag in two independent CHOattP-DLL1 (B5, C6) and CHOattP-DLL4 (B5, D3) cell lines by Western blot analysis of cell lysates with anti-Flag and anti-β-actin (for normalisation) antibodies showed similar protein levels. (C) Surface biotinylation assays demonstrated equal surface representation of DLL1 and DLL4 on CHOattP cells. (D) Notch trans-activation assays by co-culture of HeLa-N1 cells containing an RBP-Jκ:Luciferase reporter with CHOattP-DLL1 or CHOattP-DLL4 cells. All DLL1 and DLL4 clones activated Notch similarly, DLL4 being a slightly more efficient activator (compare with similar experiment in S6A and S6G Fig). (E) Notch trans-activation and cis-inhibition assays by culturing HeLa-N1 cells untransfected or transiently transfected with Dll1 or Dll4 expression constructs with or without CHOattP or CHOattP-DLL1 cells as indicated (a-c). Co-culture conditions a, b and c correspond to Luciferase measurements a’, b’ and c’, respectively. Results show cis-inhibition by DLL4 but not DLL1; for details see main text. (F)trans-Activation assays (a) without and (b) with NOTCH1 receptor expression in the signal sending CHO cell to test if NOTCH1 cis-inhibits the ligand activity of DLL1 or DLL4. No cis-inhibitory effect on either ligand was observed (columns a‘ and b‘ correspond to assay conditions a and b, respectively). (G)trans-Activation and cis-inhibition assays using chimeric DLL1-DLL4 proteins (G top; depicted as red and blue striped bars in a-c). HeLa-N1 cells were transiently transfected with no or DLL4-DLL1ECD or DLL1-DLL4ECD expression constructs and cultured as indicated (a-c). Under all three conditions, a strong cis-inhibitory activity was detected only for DLL1-DLL4ECD (columns a‘, b’ and c‘ correspond to schemas a, b and c, respectively). Error bars represent SEM; ns, not significant; *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001.
Mentions: To investigate the functional difference between DLL1 and DLL4 in vitro, we performed co-culture experiments by mixing cells expressing NOTCH1 receptor or DLL ligands and measured Notch activation with a reporter in the receptor-expressing cells. Specifically, we used HeLa cells that express both the NOTCH1 receptor (stable HeLa-N1 cells; [10]) and a transient Notch activity reporter based on an RBP-Jk promoter-driven Luciferase [56] with CHO cells stably expressing Flag-tagged DLL1 or DLL4 ligands. To ensure comparability of results, we integrated single copies of Dll1 or Dll4 ORFs under the control of the CMV promoter into the identical genomic locus of CHO cells by adopting a site-directed attP/attB recombination system (Fig 5A top; S4 Fig; [57]). We established CHO cells with a pre-inserted, randomly integrated single attP site (termed CHOattP; uniqueness of this attP site was confirmed by Southern blot analysis; S4A and S4B Fig) and recombined Dll1 or Dll4 ORFs into this site (cell lines termed CHOattP-DLL1 and CHOattP-DLL4; Fig 5A bottom left). Consistent with the expression from the same genomic locus, independent CHOattP-DLL1 (B5, C6) and CHOattP-DLL4 (B5, D3) clones expressed DLL1 and DLL4 protein at similar levels (Fig 5B; n = 4 lysates of each clone; S5A Fig, S3 Table, S5B Fig, S4 Table) and cell surface representation of DLL1 and DLL4 was similar in all lines (~40%; Fig 5C; n≥3 biotinylation assays; S5C and S5D Fig, S5 Table). Likewise, half-lives of DLL1 and DLL4 proteins were similar, DLL4 being slightly more stable (S5E and S5F Fig).

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