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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

Mesodermally expressed CAG:DLL1 but not CAG:DLL4 functionally replaces endogenous DLL1 during somitogenesis.(A) Structure of unrecombined and recombined (bottom) pMP8.CAG-Stop/Dll vector for integration of Cre-inducible expression constructs into the Hprt locus. 5‘hom and 3‘hom, 5’ and 3’ homology regions from the Hprt gene for homologous recombination; ex (grey boxes), HPRT exons; CAG prom, CAG promoter to drive transgene expression; neor, neomycin phosphotransferase; pA, polyadenylation signal; Dll1/4–Venus, Dll1 or Dll4 ORF–joined to the reporter gene Venus by an internal ribosomal entry site (IRES); hHPRT prom, human HPRT promoter; light/dark grey triangles, loxP/loxM sites (in “flip excision” orientation); “Cre” arrow, Cre-mediated recombination. (B) Venus reporter expression in E8.5 CAG:Dll1 and CAG:Dll4 embryos indicated ubiquitous transgene activation after ZP3:Cre-mediated recombination. As expected, overall fluorescence in female embryos (a,c) was weaker than in male embryos (b,d) due to random X-chromosome inactivation. Numbers of embryos analysed are given in bottom right corner. (C) Quantification of Venus protein (CAG:Dll1 set to one) by Western blot analysis of embryo lysates with anti-GFP antibodies (and anti-β-actin antibodies for normalisation) showed similar expression levels. (D) For direct comparison of DLL protein levels, we also integrated single copies of Dll1 and Dll4 labelled with C-terminal HA-tags following the strategy in (A) using recombined (active) constructs for electroporation of embryonic stem (ES) cells. Western blot analysis of three ES cell clones expressing either of these transgenes using anti-HA antibodies confirmed similar expression levels with expected mild clonal variations (a); means of all three CAG:Dll1-HA and all three CAG:Dll4-HA clones are shown in (b). (E) Cranial-caudal somite patterning visualised by whole mount in situ hybridisation of E9.5 embryos with an Uncx4.1 probe showed an extensive rescue of somitogenesis plus ectopic Notch activation by CAG:DLL1 (b,c) but no appreciable rescue of somitogenesis by CAG:DLL4 (e,f). Insets in Ea-c and Ee-g show higher magnifications of the regions as indicated. Error bars represent standard error of the mean (SEM); ns, not significant; *, P<0.05; **, P<0.01.
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pgen.1005328.g001: Mesodermally expressed CAG:DLL1 but not CAG:DLL4 functionally replaces endogenous DLL1 during somitogenesis.(A) Structure of unrecombined and recombined (bottom) pMP8.CAG-Stop/Dll vector for integration of Cre-inducible expression constructs into the Hprt locus. 5‘hom and 3‘hom, 5’ and 3’ homology regions from the Hprt gene for homologous recombination; ex (grey boxes), HPRT exons; CAG prom, CAG promoter to drive transgene expression; neor, neomycin phosphotransferase; pA, polyadenylation signal; Dll1/4–Venus, Dll1 or Dll4 ORF–joined to the reporter gene Venus by an internal ribosomal entry site (IRES); hHPRT prom, human HPRT promoter; light/dark grey triangles, loxP/loxM sites (in “flip excision” orientation); “Cre” arrow, Cre-mediated recombination. (B) Venus reporter expression in E8.5 CAG:Dll1 and CAG:Dll4 embryos indicated ubiquitous transgene activation after ZP3:Cre-mediated recombination. As expected, overall fluorescence in female embryos (a,c) was weaker than in male embryos (b,d) due to random X-chromosome inactivation. Numbers of embryos analysed are given in bottom right corner. (C) Quantification of Venus protein (CAG:Dll1 set to one) by Western blot analysis of embryo lysates with anti-GFP antibodies (and anti-β-actin antibodies for normalisation) showed similar expression levels. (D) For direct comparison of DLL protein levels, we also integrated single copies of Dll1 and Dll4 labelled with C-terminal HA-tags following the strategy in (A) using recombined (active) constructs for electroporation of embryonic stem (ES) cells. Western blot analysis of three ES cell clones expressing either of these transgenes using anti-HA antibodies confirmed similar expression levels with expected mild clonal variations (a); means of all three CAG:Dll1-HA and all three CAG:Dll4-HA clones are shown in (b). (E) Cranial-caudal somite patterning visualised by whole mount in situ hybridisation of E9.5 embryos with an Uncx4.1 probe showed an extensive rescue of somitogenesis plus ectopic Notch activation by CAG:DLL1 (b,c) but no appreciable rescue of somitogenesis by CAG:DLL4 (e,f). Insets in Ea-c and Ee-g show higher magnifications of the regions as indicated. Error bars represent standard error of the mean (SEM); ns, not significant; *, P<0.05; **, P<0.01.

Mentions: In order to directly compare the activities of DLL1 and DLL4 in vivo, we generated mice that conditionally express either Dll1 or Dll4 under the CAG promoter from a single-copy transgene insertion in the same genomic locus. We employed an established system for integration of Cre-inducible expression constructs into the Hprt locus, the pMP8.CAG-Stop vector (Fig 1A; [41,42]). The unrecombined pMP8.CAG-Stop construct expresses neomycin phosphotransferase (neor) from the CAG promoter. Cre-mediated recombination of two loxP sites and two mutant loxP2272 (loxM) sites [43] flips the gene of interest and excises neor so that the recombined construct expresses the gene of interest from the CAG promoter. 5’ and 3’ homology regions from the Hprt gene enable homologous recombination of pMP8 constructs into the Hprt locus [44]. We cloned the Dll1 and Dll4 open reading frames into the pMP8.CAG-Stop vector, introduced both unrecombined (i.e. neor expressing) constructs into Hprt-deficient E14TG2a ES cells and used homologous recombinant clones to produce transgenic mice with Cre-inducible Dll1 or Dll4 (alleles termed CAG:Dll1 and CAG:Dll4).


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)

Mesodermally expressed CAG:DLL1 but not CAG:DLL4 functionally replaces endogenous DLL1 during somitogenesis.(A) Structure of unrecombined and recombined (bottom) pMP8.CAG-Stop/Dll vector for integration of Cre-inducible expression constructs into the Hprt locus. 5‘hom and 3‘hom, 5’ and 3’ homology regions from the Hprt gene for homologous recombination; ex (grey boxes), HPRT exons; CAG prom, CAG promoter to drive transgene expression; neor, neomycin phosphotransferase; pA, polyadenylation signal; Dll1/4–Venus, Dll1 or Dll4 ORF–joined to the reporter gene Venus by an internal ribosomal entry site (IRES); hHPRT prom, human HPRT promoter; light/dark grey triangles, loxP/loxM sites (in “flip excision” orientation); “Cre” arrow, Cre-mediated recombination. (B) Venus reporter expression in E8.5 CAG:Dll1 and CAG:Dll4 embryos indicated ubiquitous transgene activation after ZP3:Cre-mediated recombination. As expected, overall fluorescence in female embryos (a,c) was weaker than in male embryos (b,d) due to random X-chromosome inactivation. Numbers of embryos analysed are given in bottom right corner. (C) Quantification of Venus protein (CAG:Dll1 set to one) by Western blot analysis of embryo lysates with anti-GFP antibodies (and anti-β-actin antibodies for normalisation) showed similar expression levels. (D) For direct comparison of DLL protein levels, we also integrated single copies of Dll1 and Dll4 labelled with C-terminal HA-tags following the strategy in (A) using recombined (active) constructs for electroporation of embryonic stem (ES) cells. Western blot analysis of three ES cell clones expressing either of these transgenes using anti-HA antibodies confirmed similar expression levels with expected mild clonal variations (a); means of all three CAG:Dll1-HA and all three CAG:Dll4-HA clones are shown in (b). (E) Cranial-caudal somite patterning visualised by whole mount in situ hybridisation of E9.5 embryos with an Uncx4.1 probe showed an extensive rescue of somitogenesis plus ectopic Notch activation by CAG:DLL1 (b,c) but no appreciable rescue of somitogenesis by CAG:DLL4 (e,f). Insets in Ea-c and Ee-g show higher magnifications of the regions as indicated. Error bars represent standard error of the mean (SEM); ns, not significant; *, P<0.05; **, P<0.01.
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pgen.1005328.g001: Mesodermally expressed CAG:DLL1 but not CAG:DLL4 functionally replaces endogenous DLL1 during somitogenesis.(A) Structure of unrecombined and recombined (bottom) pMP8.CAG-Stop/Dll vector for integration of Cre-inducible expression constructs into the Hprt locus. 5‘hom and 3‘hom, 5’ and 3’ homology regions from the Hprt gene for homologous recombination; ex (grey boxes), HPRT exons; CAG prom, CAG promoter to drive transgene expression; neor, neomycin phosphotransferase; pA, polyadenylation signal; Dll1/4–Venus, Dll1 or Dll4 ORF–joined to the reporter gene Venus by an internal ribosomal entry site (IRES); hHPRT prom, human HPRT promoter; light/dark grey triangles, loxP/loxM sites (in “flip excision” orientation); “Cre” arrow, Cre-mediated recombination. (B) Venus reporter expression in E8.5 CAG:Dll1 and CAG:Dll4 embryos indicated ubiquitous transgene activation after ZP3:Cre-mediated recombination. As expected, overall fluorescence in female embryos (a,c) was weaker than in male embryos (b,d) due to random X-chromosome inactivation. Numbers of embryos analysed are given in bottom right corner. (C) Quantification of Venus protein (CAG:Dll1 set to one) by Western blot analysis of embryo lysates with anti-GFP antibodies (and anti-β-actin antibodies for normalisation) showed similar expression levels. (D) For direct comparison of DLL protein levels, we also integrated single copies of Dll1 and Dll4 labelled with C-terminal HA-tags following the strategy in (A) using recombined (active) constructs for electroporation of embryonic stem (ES) cells. Western blot analysis of three ES cell clones expressing either of these transgenes using anti-HA antibodies confirmed similar expression levels with expected mild clonal variations (a); means of all three CAG:Dll1-HA and all three CAG:Dll4-HA clones are shown in (b). (E) Cranial-caudal somite patterning visualised by whole mount in situ hybridisation of E9.5 embryos with an Uncx4.1 probe showed an extensive rescue of somitogenesis plus ectopic Notch activation by CAG:DLL1 (b,c) but no appreciable rescue of somitogenesis by CAG:DLL4 (e,f). Insets in Ea-c and Ee-g show higher magnifications of the regions as indicated. Error bars represent standard error of the mean (SEM); ns, not significant; *, P<0.05; **, P<0.01.
Mentions: In order to directly compare the activities of DLL1 and DLL4 in vivo, we generated mice that conditionally express either Dll1 or Dll4 under the CAG promoter from a single-copy transgene insertion in the same genomic locus. We employed an established system for integration of Cre-inducible expression constructs into the Hprt locus, the pMP8.CAG-Stop vector (Fig 1A; [41,42]). The unrecombined pMP8.CAG-Stop construct expresses neomycin phosphotransferase (neor) from the CAG promoter. Cre-mediated recombination of two loxP sites and two mutant loxP2272 (loxM) sites [43] flips the gene of interest and excises neor so that the recombined construct expresses the gene of interest from the CAG promoter. 5’ and 3’ homology regions from the Hprt gene enable homologous recombination of pMP8 constructs into the Hprt locus [44]. We cloned the Dll1 and Dll4 open reading frames into the pMP8.CAG-Stop vector, introduced both unrecombined (i.e. neor expressing) constructs into Hprt-deficient E14TG2a ES cells and used homologous recombinant clones to produce transgenic mice with Cre-inducible Dll1 or Dll4 (alleles termed CAG:Dll1 and CAG:Dll4).

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