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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

DLL4 expressed from the Dll1 locus rescues DLL1 loss-of-function in the retina.(A)Dll1  mutant retinas show epithelial disruption with formation of polarised rosettes in which the apical markers N-Cadherin (NCad, a) and ZO-1 (ZO1, b) are abnormally present at the central lumen. Ectopic proliferating progenitors, labelled with PHH3 (b, arrowheads), are located close to the apical lumen of these rosettes. (B) In contrast, the neuroepithelium of homozygous Dll1Dll1ki and Dll1Dll4ki embryos is correctly organised without rosettes, and N-Cadherin shows the normal apical localisation close to the retinal pigmented epithelium (a,b). Mitotic progenitors (PHH3+) are only detected at the apical region of the neuroepithelium (a,b arrowheads). A normal stratification of CHX10+ progenitors and P27+ differentiating neurons is also observed (c,d). (C, D) E13.5 homozygous Dll1Dll1ki and Dll1Dll4ki retinas show no significant difference in the number of ISL1+ RGCs (C) and CRABP+ amacrine cells (D). Cells immunopositive for Islet-1 and Crabp were counted and related to the total number of cells in the retina (DAPI+). Percentages are shown as mean ± SEM; ns, not significant. (E) Expression of DLL4 in homozygous Dll1Dll1ki (a,c) and in homozygous Dll1Dll4ki (b,d) E13.5 retinas as detected by an anti-DLL4 antibody. (c) and (d) are magnifications of (a) and (b), respectively. Endogenous plus transgenic DLL4 is expressed in more cells in Dll1Dll4ki/Dll4ki as compared to endogenous DLL4 expression in Dll1Dll1ki/Dll1ki while signal strength is similar. Scale bars are 50 μm in (A, B) and 100 μm in (E).
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pgen.1005328.g004: DLL4 expressed from the Dll1 locus rescues DLL1 loss-of-function in the retina.(A)Dll1 mutant retinas show epithelial disruption with formation of polarised rosettes in which the apical markers N-Cadherin (NCad, a) and ZO-1 (ZO1, b) are abnormally present at the central lumen. Ectopic proliferating progenitors, labelled with PHH3 (b, arrowheads), are located close to the apical lumen of these rosettes. (B) In contrast, the neuroepithelium of homozygous Dll1Dll1ki and Dll1Dll4ki embryos is correctly organised without rosettes, and N-Cadherin shows the normal apical localisation close to the retinal pigmented epithelium (a,b). Mitotic progenitors (PHH3+) are only detected at the apical region of the neuroepithelium (a,b arrowheads). A normal stratification of CHX10+ progenitors and P27+ differentiating neurons is also observed (c,d). (C, D) E13.5 homozygous Dll1Dll1ki and Dll1Dll4ki retinas show no significant difference in the number of ISL1+ RGCs (C) and CRABP+ amacrine cells (D). Cells immunopositive for Islet-1 and Crabp were counted and related to the total number of cells in the retina (DAPI+). Percentages are shown as mean ± SEM; ns, not significant. (E) Expression of DLL4 in homozygous Dll1Dll1ki (a,c) and in homozygous Dll1Dll4ki (b,d) E13.5 retinas as detected by an anti-DLL4 antibody. (c) and (d) are magnifications of (a) and (b), respectively. Endogenous plus transgenic DLL4 is expressed in more cells in Dll1Dll4ki/Dll4ki as compared to endogenous DLL4 expression in Dll1Dll1ki/Dll1ki while signal strength is similar. Scale bars are 50 μm in (A, B) and 100 μm in (E).

Mentions: In the embryonic neural retina, Dll1 and Dll4 are sequentially expressed and can both function to maintain proliferating progenitors, while they have different functions in retinal fate diversification [51,55]. In contrast to myogenesis, DLL4 can fully replace DLL1 function in maintaining neuronal progenitors in the embryonic retina. Whereas Dll1 mutants show a striking disruption of the retinal neuroepithelium with formation of rosettes (Fig 4A), due to premature differentiation of retinal progenitors [51], both Dll1Dll1ki/Dll1ki and Dll1Dll4ki/Dll4ki retinas have a normal neuroepithelial organisation with a clear stratification of Chx10+ progenitors and p27+ differentiating neurons (Fig 4B). Moreover, we find that similar numbers of early born retinal neurons [retinal ganglion cells (RGCs) and amacrine cells] are present in Dll1Dll1ki and Dll1Dll4ki retinas (Fig 4C and 4D; n≥4 retinal sections), confirming that DLL1 and DLL4 functions are interchangeable in regulating early retinal neurogenesis. We have further analysed DLL4 expression in Dll1Dll4ki/Dll4ki retinas and found it recapitulates the broader Dll1 expression pattern, with the transgenic protein expressed at similar levels as endogenous DLL4 in the retinal neuroepithelium (compare Fig 4Ea–4Ec with 4Eb–4Ed). Together, these results offer further evidence that the Dll4 transgene is fully functional in Dll1Dll4ki/Dll4ki embryos. The extent of the functional equivalence of DLL1 and DLL4 depends on the developmental context.


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)

DLL4 expressed from the Dll1 locus rescues DLL1 loss-of-function in the retina.(A)Dll1  mutant retinas show epithelial disruption with formation of polarised rosettes in which the apical markers N-Cadherin (NCad, a) and ZO-1 (ZO1, b) are abnormally present at the central lumen. Ectopic proliferating progenitors, labelled with PHH3 (b, arrowheads), are located close to the apical lumen of these rosettes. (B) In contrast, the neuroepithelium of homozygous Dll1Dll1ki and Dll1Dll4ki embryos is correctly organised without rosettes, and N-Cadherin shows the normal apical localisation close to the retinal pigmented epithelium (a,b). Mitotic progenitors (PHH3+) are only detected at the apical region of the neuroepithelium (a,b arrowheads). A normal stratification of CHX10+ progenitors and P27+ differentiating neurons is also observed (c,d). (C, D) E13.5 homozygous Dll1Dll1ki and Dll1Dll4ki retinas show no significant difference in the number of ISL1+ RGCs (C) and CRABP+ amacrine cells (D). Cells immunopositive for Islet-1 and Crabp were counted and related to the total number of cells in the retina (DAPI+). Percentages are shown as mean ± SEM; ns, not significant. (E) Expression of DLL4 in homozygous Dll1Dll1ki (a,c) and in homozygous Dll1Dll4ki (b,d) E13.5 retinas as detected by an anti-DLL4 antibody. (c) and (d) are magnifications of (a) and (b), respectively. Endogenous plus transgenic DLL4 is expressed in more cells in Dll1Dll4ki/Dll4ki as compared to endogenous DLL4 expression in Dll1Dll1ki/Dll1ki while signal strength is similar. Scale bars are 50 μm in (A, B) and 100 μm in (E).
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pgen.1005328.g004: DLL4 expressed from the Dll1 locus rescues DLL1 loss-of-function in the retina.(A)Dll1 mutant retinas show epithelial disruption with formation of polarised rosettes in which the apical markers N-Cadherin (NCad, a) and ZO-1 (ZO1, b) are abnormally present at the central lumen. Ectopic proliferating progenitors, labelled with PHH3 (b, arrowheads), are located close to the apical lumen of these rosettes. (B) In contrast, the neuroepithelium of homozygous Dll1Dll1ki and Dll1Dll4ki embryos is correctly organised without rosettes, and N-Cadherin shows the normal apical localisation close to the retinal pigmented epithelium (a,b). Mitotic progenitors (PHH3+) are only detected at the apical region of the neuroepithelium (a,b arrowheads). A normal stratification of CHX10+ progenitors and P27+ differentiating neurons is also observed (c,d). (C, D) E13.5 homozygous Dll1Dll1ki and Dll1Dll4ki retinas show no significant difference in the number of ISL1+ RGCs (C) and CRABP+ amacrine cells (D). Cells immunopositive for Islet-1 and Crabp were counted and related to the total number of cells in the retina (DAPI+). Percentages are shown as mean ± SEM; ns, not significant. (E) Expression of DLL4 in homozygous Dll1Dll1ki (a,c) and in homozygous Dll1Dll4ki (b,d) E13.5 retinas as detected by an anti-DLL4 antibody. (c) and (d) are magnifications of (a) and (b), respectively. Endogenous plus transgenic DLL4 is expressed in more cells in Dll1Dll4ki/Dll4ki as compared to endogenous DLL4 expression in Dll1Dll1ki/Dll1ki while signal strength is similar. Scale bars are 50 μm in (A, B) and 100 μm in (E).
Mentions: In the embryonic neural retina, Dll1 and Dll4 are sequentially expressed and can both function to maintain proliferating progenitors, while they have different functions in retinal fate diversification [51,55]. In contrast to myogenesis, DLL4 can fully replace DLL1 function in maintaining neuronal progenitors in the embryonic retina. Whereas Dll1 mutants show a striking disruption of the retinal neuroepithelium with formation of rosettes (Fig 4A), due to premature differentiation of retinal progenitors [51], both Dll1Dll1ki/Dll1ki and Dll1Dll4ki/Dll4ki retinas have a normal neuroepithelial organisation with a clear stratification of Chx10+ progenitors and p27+ differentiating neurons (Fig 4B). Moreover, we find that similar numbers of early born retinal neurons [retinal ganglion cells (RGCs) and amacrine cells] are present in Dll1Dll1ki and Dll1Dll4ki retinas (Fig 4C and 4D; n≥4 retinal sections), confirming that DLL1 and DLL4 functions are interchangeable in regulating early retinal neurogenesis. We have further analysed DLL4 expression in Dll1Dll4ki/Dll4ki retinas and found it recapitulates the broader Dll1 expression pattern, with the transgenic protein expressed at similar levels as endogenous DLL4 in the retinal neuroepithelium (compare Fig 4Ea–4Ec with 4Eb–4Ed). Together, these results offer further evidence that the Dll4 transgene is fully functional in Dll1Dll4ki/Dll4ki embryos. The extent of the functional equivalence of DLL1 and DLL4 depends on the developmental context.

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