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

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.


Introduction
The Notch signalling pathway mediates local interactions between adjacent cells and thereby regulates numerous developmental processes in a wide variety of different tissues throughout the animal kingdom [reviewed in [1][2][3][4][5][6][7].The Notch gene of Drosophila and its vertebrate homologues encode large transmembrane proteins that act as receptors at the surface of the cell.They interact with transmembrane ligand proteins on the surface of neighbouring, signal-sending cells (i.e. in trans) encoded by the Delta and Serrate (called Jagged in vertebrates) genes.Upon ligand binding, the intracellular domain of Notch (NICD) is proteolytically released, translocates to the nucleus, interacts with the transcriptional regulator Suppressor of Hairless ([Su(H)]; CSL proteins in vertebrates) and activates the transcription of downstream target genes [8][9][10][11][12][13][14].Ligands coexpressed with the Notch receptor in signal-receiving cells (i.e. in cis) are capable of interacting with Notch and attenuate the signal strength [15-17, reviewed in 18].
Vertebrates possess several Notch receptors and ligands.The mouse genome encodes four Notch (NOTCH1-4), three Delta (DLL1, DLL3 and DLL4) and two Jagged (JAG1 and JAG2) proteins.Among the DLL proteins, only DLL1 and DLL4 function as Notch-activating ligands [19][20][21].As paralogues, DLL1 and DLL4 are similar in sequence (47% identical plus 14% similar amino acids), size and domain structure [22].Both contain a DSL domain, which is essential for the interaction with Notch [23,24], as well as eight EGF-like repeats in their extracellular domain and have a short intracellular domain with a C-terminal PDZ binding motif.Dll1 and Dll4 are expressed both in discrete and overlapping patterns during embryonic development and in adult tissues of the mouse.In shared expression domains, the two ligands have redundant or different functions depending on the developmental context.An example for full redundancy is the maintenance of the crypt progenitor pool in the adult small intestine.Dll1 and Dll4 are coexpressed in crypt cells [25,26] and individual inactivation of either ligand has no effect on the crypt progenitor cell pool.However, simultaneous deletion of Dll1 and Dll4 leads to a complete loss of the proliferative crypt compartment and intestinal stem cells [27].
Conversely, in foetal arteries where both ligands are expressed in the vascular endothelium [26,28,29] inactivation of Dll1 causes loss of NOTCH1 activation despite the presence of DLL4 [29] suggesting that DLL4 cannot compensate for the loss of DLL1 in fetal endothelial cells.In the adult thymus, Dll1 and Dll4 are both expressed in thymic epithelial cells [26,30].Here, DLL4 is the essential Notch ligand required for T-lymphopoiesis [31] and T cell development is unaltered in mice lacking DLL1 in the thymic epithelium [32] suggesting that in this context DLL1 and DLL4 are functionally distinct.This conclusion is supported by in vitro studies showing that DLL1 and DLL4 differ with respect to their binding avidity to Notch receptors on thymocytes and to the steady-state cell surface levels required to induce T cell development, DLL4 being the more effective ligand [33,34] as well as by biochemical studies indicating a 10-fold higher Notch binding affinity of DLL4 than DLL1 [19].Furthermore, DLL4 but not DLL1 can induce a fate switch in skeletal myoblasts and induce pericyte markers [35].Collectively, these individual reports of context-dependent redundant and distinct functions of coexpressed DLL1 and DLL4 raise the questions of why DLL1 and DLL4 act equally in some processes but differently in others, which mechanism or factor causes their function to vary and whether they are similar enough to replace each other in domains where only one of both DLL ligands is endogenously expressed.
In early mouse embryos, expression of Dll1 and Dll4 is largely non-overlapping.Dll1 is expressed in the paraxial mesoderm beginning at E7.5, in the central nervous system from E9 onwards and later on, at E13.5, in arterial endothelial cells [29,36].Deletion of Dll1 disrupts somite patterning and causes premature myogenic differentiation, severe haemorrhages and embryonic death after E11 [37,38].Dll4 is expressed in the vascular endothelium of arteries beginning at E8 [39] but not in the somite-generating presomitic mesoderm, somites or differentiating myoblasts.Inactivation of DLL4 results in severe vascular defects leading to embryonic death prior to E10.5 [39,40].
Here, we address the functional equivalence of DLL1 and DLL4 in vivo and in vitro.We analyse Notch signalling in mice that conditionally overexpress DLL1 or DLL4 on a Dll1 null genetic background and in mice in which Dll1 is replaced by Dll4, focussing on young embryos in which both Notch ligands have discrete endogenous expression domains.We show that DLL4 cannot replace DLL1 during somite segmentation but can partially replace DLL1 during myogenesis and fully replace DLL1 during maintenance of retinal progenitors.Cell culture assays that measure Notch activation by DLL1 or DLL4 demonstrate that DLL4 trans-activates Notch signalling similarly to DLL1 but cis-inhibits Notch signalling much more efficiently than DLL1, partly overruling the activation by interactions in trans.Consistent with these in vitro data, we observe dominant effects on segmentation by DLL4 ectopically expressed in the presomitic mesoderm (PSM).We propose that differential Notch cis-inhibition by DLL1 and DLL4 contributes to the observed tissue-dependent functional divergence of both paralogues, perhaps in combination with other factors not tested in this study.

Mesodermal expression of DLL1 but not DLL4 rescues the Dll1 knockout somitogenesis phenotype
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 (neo r ) 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 neo r 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.neo r 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).
To check activation of DLL1 and DLL4 in embryos, we induced ubiquitous expression of the CAG:Dll1 and CAG:Dll4 transgene by mating our mice with mice carrying a ZP3:Cre transgene that causes site-specific recombination during oogenesis [45].We crossed CAG:Dll1; ZP3:Cre and CAG:Dll4;ZP3:Cre females with wildtype males to obtain embryos that overexpress Dll1 or Dll4 from the zygote stage on.The transgenes are transcribed bicistronically with an IRES-Venus (Fig 1A) whose expression marks cells in which Cre-recombination activated the transgene.As Hprt is located on the X chromosome, hemizygous male embryos expressed Venus ubiquitously whereas heterozygous female embryos showed mosaic expression due to random X-inactivation (Fig 1B).Analysis of embryo lysates on a Western blot with anti-GFP antibodies demonstrated CAG:Dll1 and -4 transgene activation at similar levels As expected, inactivation of Dll1 throughout the mesoderm resulted in severe somite patterning defects characterised by loss of Uncx4.1 expression (Fig 1Ed ), a marker for caudal somite compartments [47,48] whose expression depends on Notch activation [46].Expression of CAG:DLL1 in such Dll1-deficient embryos restored robust, largely regularly striped expression of Uncx4.1, which expanded into cranial somite compartments in most axial regions and particularly in hemizygous male embryos (Fig 1Eb and 1Ec).This rescue of somitogenesis demonstrates that expression of CAG:DLL1 (from the Hprt locus) is sufficient to substitute for the loss of endogenous DLL1; cranial expansion of Uncx4.1 is reminiscent of ectopic Notch activity [46].
In contrast, expression of CAG:DLL4 in Dll1-deficient embryos restored only very weak and irregular expression of Uncx4.1 and resembles Dll1 loxP/loxP ;T(s):Cre embryos without CAG: DLL1 overexpression (Fig 1Ee and 1Ef).Only two out of 16 embryos of this genotype displayed regular Uncx4.1 expression in the cranial most somites, which might reflect residual DLL1 activity perhaps due to delayed excision of endogenous Dll1 (Fig 1Eg).The extensively defective segmentation in Dll1 loxP/loxP ;T(s):Cre embryos with CAG:DLL4 overexpression directly shows a functional difference between DLL1 and DLL4 during early embryogenesis: DLL4 is not able to take over DLL1 function in the paraxial mesoderm during somite formation.Weak and irregular Uncx4.1 expression in some of these embryos suggest Notch activation at low levels.

Mice expressing DLL4 in place of DLL1 from the Dll1 locus reveal divergent function during somitogenesis
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 Dll1 Dll4ki .As a control, we used the analogous knock-in of a Dll1 mini gene into the Dll1 locus (Fig 2A bottom; Dll1 tm2Gos , here referred to as Dll1 Dll1ki ), 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 Dll1 Dll1ki mice were viable and fertile and appeared phenotypically normal indicating that the Dll1 mini gene can functionally substitute the endogenous Dll1 gene [37].Heterozygous Dll1 Dll4ki/+ mice (containing one endogenous copy of Dll1 and one copy of Dll4ki) were viable and fertile and showed no obvious phenotype except for kinky tails (Fig 2Ba -2Bc, arrow; penetrance 89%; n = 48), a phenotype indicative of irregular somitogenesis rarely observed in Dll1 Dll1ki homozygotes or Dll1 null (Dll1 lacZ ) heterozygotes (penetrance 15%; n = 23 and 53, respectively; Fig 2Ba'-2Bc').In contrast to homozygous Dll1 Dll1ki , no homozygous Dll1 Dll4ki mice were obtained after birth.At E15.5, Dll1 Dll4ki homozygotes exhibited short body axes, truncated tails and were oedematic (Fig 2C; arrow points at tip of tail) resembling foetuses with severely reduced DLL1 function [37].
Correct expression of Dll4 in the presomitic mesoderm (PSM) of Dll1 Dll4ki embryos was confirmed by in situ hybridisation using probes specific for the Dll4 ORF or the 3'UTR (Dll1  [50,51].Northern blot analysis of Dll1 Dll1ki and Dll1 Dll4ki homozygous embryos indicated equal levels of transcription of the transgenes (Fig 2E).In Dll1 Dll4ki / Dll4ki embryos, ectopic DLL4 protein was detected at the plasma membrane of PSM cells (Fig 2Fa -2Fc).Likewise, DLL1 protein was detected at the surface of PSM cells in homozygous Dll1 Dll1ki embryos (Fig 2Fj -2Fl), confirming that DLL4 and DLL1 protein is generated from their mini genes and targeted to the plasma membrane in vivo.Taken together, these data show that Dll1 Dll4ki mice indeed express Dll4 instead of Dll1 from the Dll1 locus at comparable levels and confirm our previous observation that DLL4 is unable to support proper mouse development in the absence of endogenous DLL1.
Cranial-caudal somite patterning critically depends on DLL1-mediated Notch signalling [38,46,52].We analysed if DLL4 can functionally replace DLL1 in this process in homozygous Dll1 Dll4ki embryos.Unlike embryos that contained at least one wildtype or Dll1 Dll1ki allele, homozygous Dll1 Dll4ki embryos displayed severely reduced and irregular Uncx4.1 expression (Fig 3A ), which indicates disrupted somite patterning and reduced Notch activity in the PSM due to the inability of DLL4 to replace DLL1.Consistent with defective somite formation and the shortened body axis observed in E15.5 foetuses, Dll1 Dll4ki/Dll4ki axial skeletons were severely disorganised (Fig 3B).Therefore, expression of DLL4 from the Dll1 locus does not cause a significant rescue of the Dll1 somitogenesis phenotype.

DLL4 can partially substitute for DLL1 during myogenesis and fully replace DLL1 during early retinal development
Processes other than somitogenesis in the developing embryo that depend on DLL1-Notch signalling include myogenesis [37] and retinal development [51].Embryos lacking DLL1 display excessive differentiation of myoblasts, which exhausts the progenitor pool and leads to severely reduced or absent skeletal muscles [37].Homozygous E9.5 Dll1 Dll4ki embryos showed transient upregulation of the myocyte marker Myogenin

DLL1 and DLL4 activate Notch similarly in vitro
To investigate the functional difference between DLL1 and DLL4 in vitro, we performed coculture 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 CHO attP ; uniqueness of this attP site was confirmed by Southern blot analysis; S4A and S4B  Co-culture of HeLa-N1 with either CHO attP-DLL1 or CHO attP-DLL4 (schematically shown in Fig 5A bottom) led to a >10-fold increase of Notch activity as compared to co-cultures of HeLa-N1 with CHO attP cells that did not express transgenic DLL1 or DLL4 (Fig 5D; n = 3) confirming that all transgenes were functional.DLL4 trended to activate Notch more strongly than DLL1 (including clone CHO attP-DLL4 B5 whose protein level was slightly reduced in Fig 5B ); the difference between individual clones was not statistically significant in these experiments and partly significant in similar experiments with other clones (S6A and S6G Fig) .Next, we tested whether coexpression of further factors (LFNG, JAG1) in our cell culture system differently alters Notch activation by DLL1 or DLL4 and thereby provides a plausible explanation for the distinct phenotypes.The glycosyltransferase LUNATIC FRINGE (LFNG), which is expressed in the PSM, is able to modify NOTCH in the trans-Golgi [58,59] and thereby modulates receptor activation.The Notch ligand JAG1 is expressed in forming somites [60,61] and can act as a competitive inhibitor of DLL ligands [62,63].We performed co-culture assays with HeLa-N1 cells transiently expressing LFNG-HA (S6A-S6C Also, different glycosylation patterns of the ligands' extracellular domain could contribute to differences in their activity.To test this possible influence, we treated co-cultures with tunicamycin to prevent N-glycosylation.Blocking N-glycosylation reduced the activity of DLL4 in cultured cells significantly, but not below DLL1 activity (S6G and S6H Fig) , suggesting that distinct N-glycosylation is an unlikely cause for the observed differences between both ligands.Collectively, our results do not reveal a difference in the trans-activation potential of DLL1 and DLL4 that could explain the different segmentation phenotypes of our transgenic DLL1-or DLL4-expressing mice.In summary, our cis-inhibition assays (Fig 5E) reveal a functional difference between DLL1 and DLL4 that was not evident in the trans-activation assays (Fig 5D and 5F): DLL4, but not DLL1, is a potent cis-inhibitor of NOTCH1 and cis-inhibition by DLL4 can significantly reduce Notch activation.Our in vitro results are consistent with our in vivo data: they can explain both why DLL4 appears to be a weaker activator of Notch signalling than DLL1 during somitogenesis in our transgenic mice and why transgenic DLL4 has a dominant effect on segmentation in Dll1 Dll4ki/+ mice (see Discussion and Fig 6).We propose that in the PSM, DLL1 is a more efficient net activator of Notch than (ectopic) DLL4 because it does not efficiently cis-inhibit Notch.

The cis-inhibitory potential of DLL4 is mediated by its extracellular domain
In order to identify the protein domain that mediates cis-inhibition by DLL4, we cloned chimeric show that cis-inhibition is mediated by the extracellular domain of DLL4.This observation is consistent with studies that showed that the DSL domain as well as EGF repeats 4-6 of Serrate are essential for cis-inhibition in Drosophila although these EGF repeats are not well conserved between Serrate and Delta ligands [24,65,66].Analysis of Notch activation by chimeric proteins in which smaller domains of the extracellular regions are swapped will help to precisely map the cis-inhibitory domain in DLL4.

Discussion
The presence of several Notch receptors and ligands in mammals offers a multitude of possible receptor-ligand interactions; whether different combinations of receptor and ligand qualitatively or quantitatively vary in their signalling output is largely unknown.In this study, we focus on the mouse Notch ligands DLL1 and DLL4 and find functional differences in vivo, which are particularly apparent in the PSM: DLL4 cannot replace DLL1 during axial segmentation, and a striking dominant segmentation phenotype in Dll1 Dll4ki/+ mice hints towards an inhibitory function of ectopically expressed DLL4 in the PSM.We examined the possibility that differential cis-inhibition contributes to the phenotype and our in vitro Notch activation data are indeed consistent with this possibility (Fig 6), but do not exclude that other factors may contribute (see below).

Direct comparison of DLL1 and DLL4 equally expressed in embryos uncover context-dependent differences in their ability to activate Notch
Mesodermal expression of DLL1 and DLL4 from the Hprt locus on a Dll1 mutant background caused different phenotypes providing first hints that DLL1 and DLL4 are functionally In our mouse models, we expressed untagged Dll1 and Dll4 transgenes to avoid alteration of protein function by the tag.As a consequence, we were unable to directly compare DLL1 and DLL4 levels in vivo and therefore cannot exclude small differences that may have contributed in part to the observed phenotype; strong differences are not indicated in the controls mentioned above.
Also, it is very unlikely that DLL1 or DLL4 have functions other than interacting with and activating Notch receptors.Although it has been previously suggested that the intracellular domain of DLL1 may influence gene transcription in the signal sending cell [67,68], we were unable to reproduce these in vitro results and showed that overexpression of the intracellular domain of DLL1 does not cause a phenotype in mice [42].Collectively, the distinct ability to cis-inhibit Notch is a plausible explanation for the context-dependent DLL1-DLL4-divergence.

Distinct cis-inhibitory capacity of DLL1 and DLL4 in vitro
The ability of vertebrate DLL homologues to cis-inhibit Notch has been suggested before: overexpression of truncated DLL1 proteins lacking the intracellular domain in Xenopus, chicken and mouse embryos show dominant-negative effects on Notch signalling that are likely to be caused by cis-inhibition of Notch [53,69,70].In primary human keratinocyte cultures, expression of DLL1 (and truncated DLL1 T ) renders cells unresponsive to Delta signals from neighbouring cells and controls differentiation of stem cells [71].Our data show for the first time that DLL4 is a strong cis-inhibitor of Notch signalling, far stronger than DLL1.We have examined cis-inhibition in various types of cultures, in NOTCH-and DLL-expressing HeLa cells with and without co-culture of empty or DLL-expressing CHO cells and with chimeric DLL1-4 proteins (Fig 5E and 5G).Furthermore, we have tested cis-inhibition of DLL1 and DLL4 ligands by NOTCH1 (Fig 5F).All those assays consistently show a strong reduction of Notch signalling by DLL4 when coexpressed with NOTCH1.
In our assays, DLL1 had no obvious cis-inhibitory effect (Fig 5Eb'; n = 6), which differs from earlier reports showing that vertebrate DLL1 proteins can cis-inhibit NOTCH1 [20,[72][73][74].This is likely due to different assay conditions: in these previous studies, DLL1 was derived from different vertebrate species or differently tagged, or different cell systems or higher ligand concentrations were used.In studies in which cis-inhibition of Notch by Delta and Serrate was compared, Delta displayed a relatively weaker cis-inhibitory potential [75,76].
The ability for strong cis-inhibition resides in the extracellular domain of DLL4 (Fig 5G) that physically interacts with the Notch extracellular domain.Possible causes for the higher cis-inhibitory potency of DLL4 as compared to DLL1 include a potentially higher Notch cisbinding affinity of DLL4 as determined for the trans-interaction in vitro [34] or different glycosylation patterns in the extracellular domains of DLL1 and DLL4 (DLL4 contains an additional O-fucosylation site in EGF5 and four additional N-glycosylation sites, three of which reside in the N-terminal domain, which is essential for Notch activation; e.g.[19]; sites predicted by www.cbs.dtu.dk/services/NetOGlyc).

cis-inhibition by DLL4 in vivo
Our in vitro findings provide a possible explanation why DLL1 supports regular somite formation whereas DLL4 with its reduced net Notch activation potential is unable to do so.Heterozygous Dll1 Dll4ki/+ mice consistently exhibit kinky tails and irregular vertebrae (Figs 2B and 3Bd; S3 Fig) despite the presence of one wildtype Dll1 allele, which should be able to support regular somitogenesis [53].This finding strongly supports an in vivo inhibitory effect of DLL4 in the PSM, in which Dll4 is ectopically expressed at physiological levels (similar to the endogenous Dll1 levels; Fig 2E).Skeletal malformations observed in Dll1 Dll4ki/+ mice are distinct from phenotypes observed upon mild overexpression of Dll1 in the paraxial mesoderm that include fused or split vertebral bodies and reduction of costal heads of ribs [77].This supports the view that cis-inhibitory DLL4 acts in a dominant-negative manner partially overruling Notch activation by wildtype DLL1 causing axial skeleton defects in Dll1 Dll4ki/+ mice, similar to the effect of a truncated dominant-negative form of DLL1 expressed in the paraxial mesoderm [53].An alternative explanation for the dominant segmentation effect in heterozygous Dll1 Dll4ki/+ mice could be a competition between DLL4 and DLL1 for NOTCH binding sites with DLL4 binding NOTCH more efficiently but activating it less efficiently than DLL1; although DLL1 has not been shown to be a more potent activator of NOTCH in vitro (Fig 5D ; S6A, S6D and S6G Fig; [33,34]) we cannot exclude that this is the case in certain cellular contexts.
cis-Inhibition has been demonstrated to play a physiological role during fly development at the dorso-ventral border of the wing imaginal disc [15,16] and in photoreceptor precursors of the eye [17].In vertebrates, the occurrence of cis-inhibition under physiological conditions is less clear but probable (see previous section).We did not observe apparent phenotypes in Dll1 Dll4ki/+ mice that indicate dominant-negative effects of DLL4 outside the PSM.However, we hypothesise that cis-inhibition may occur in the foetal arterial endothelium, where DLL1, DLL4 and NOTCH1 are coexpressed and where loss of DLL1 abolishes NOTCH1 activation [29], possibly due to cis-inhibition by DLL4.
The PSM is particularly well suited to test the functionality of Notch ligands in vivo because DLL1 is the only activating ligand endogenously expressed in this tissue [78] and Dll4 mutants have no somitogenesis phenotype [39,40], so the analysis of Notch signalling is not complicated by the presence of several activators or confounded by composite phenotypes.However, two receptors, NOTCH1 and NOTCH2, are expressed in the PSM and may differ in their response to DLL1 or DLL4 binding.
The situation in myoblasts and other tissues is less clear.Outside the PSM, receptor and ligand expression typically exclude each other so that cis-inhibition can occur only during the short process in which the fate as receptor-or ligand-expressing cells is established [76,79,80].That way, cis-inhibition may also be responsible for differences between DLL1 and DLL4 observed during myogenesis (Fig 3D -3F).Other reasons may contribute to or cause these differences: Firstly, further Notch receptors and ligands are expressed during myogenesis [81].The contribution of individual Notch receptors to myogenesis is unknown but their function could vary [82].Also, different ligands, including DLL1 and DLL4, have been shown to activate different Notch targets depending on the cell type in vitro [83,84].A future thorough analysis of the functional divergence between DLL1 and DLL4 in myoblasts should aim at identifying the involved receptors and modulators (perhaps by in vitro analyses of myogenic or mesodermal progenitor cells including knock-down of individual factors) in order to understand the mechanisms underlying the observed phenotype.
Secondly, other processes may cause the divergence, e.g.modification of the ligands or receptors by glycosylation (may also play a role in the PSM).Activation of Notch by its ligands can be modulated by Fringe proteins.While glycosylation of Notch by LFNG enhances interaction with DLL1 in C2C12 cells [85] and with DLL4 in T cells in vitro [86], it appears to attenuate Notch signalling in the PSM [64,87].However, we did not observe any shortcomings of DLL4 in the ability to trans-activate NOTCH1 compared to DLL1 when LFNG was present in the receptor-presenting cell (S6A-S6C Fig) .The trans-activation potential of DLL1 and DLL4 could vary under certain conditions in vivo, perhaps depending on the glycosylation status, although our in vitro assays did not reveal any difference.Finally, the different extent of the functional difference between DLL1 and DLL4 observed in the PSM and during myogenesis may reflect the fact that mild changes of DLL1 activity affect the delicate Notch signalling in the PSM more readily than outside the PSM because somite patterning appears to be particularly sensitive to reduced Notch activity [88].
In conclusion, our genetic studies revealed a context-dependent functional divergence of the NOTCH ligands DLL1 and DLL4 in mice and provide a basis for a more extensive mechanistic analysis of this divergence in future studies.These will identify the relevant protein domain(s) and biochemical parameters and contribute to our understanding how different combinations of receptors and ligands determine the outcome of Notch signalling.CMV-Dll4mini-pA.The Dll4ki mini gene was released from the Dll4ki targeting construct with SacI/XbaI and cloned into expression vector pTracer-CMV (Invitrogen).Dll1 and Dll4 ORFs (Flag-tagged) were cloned into the multiple cloning site of pTracer-CMV and used as controls.

Cloning of constructs
pHZ-attP-Jag1Myc.A DNA fragment containing Jagged1-Myc and flanking frt sites was synthesised (Life technologies) and inserted into the MluI site of the pHZ-attP vector [57].

Generation and husbandry of transgenic mice
Ethics statement.All animal experiments were performed according to the German rules and regulations (Tierschutzgesetz) and approved by the ethics committee of Lower Saxony for care and use of laboratory animals LAVES (Niedersächsisches Landesamt für Verbraucherschutz und Lebensmittelsicherheit). Mice were housed in the central animal facility of Hannover Medical School (ZTL) and were maintained as approved by the responsible Veterinary Officer of the City of Hannover.Animal welfare was supervised and approved by the Institutional Animal Welfare Officer (Tierschutzbeauftragter).

Analyses of gene expression patterns and phenotypes
Whole mount in situ hybridisation.Embryos were collected in ice cold phosphate buffered saline (PBS) and fixed over night in 4% formaldehyde/PBS at 4°C.Hybridisation was performed following standard procedures [91] with digoxigenin labelled cDNA probes comprising the whole ORFs of Dll1 or Dll4, Dll1 exon 11, ~1kb of the 5' region of Myogenin cDNA [92] and Uncx4.1 [47].Photos were taken with the Leica Z6 APO microscope and Leica Fire-Cam software.
Antibody staining.E15.5 embryos were fixed over night in 4% formaldehyde/PBS at 4°C, dehydrated in methanol and 2-propanol, embedded in paraffin and sectioned sagittally (10 μm).Antigene unmasking was performed by cooking sections 20 min in 10 mM Tris pH9.5/1 mM EDTA.Sections were blocked in 1% BSA/2% goat serum 1h at room temperature, incubated with a monoclonal mouse anti-myosin antibody (skeletal, fast, My32; dilution 1:250; Sigma-Aldrich) in blocking solution over night at 4°C, incubated with secondary goat biotinylated anti-mouse antibody (BA 9200; 1:200; Vector Laboratories) in blocking solution 1 hour at RT and stained with VECTASTAIN Elite ABC Kit and DAB (Vector Laboratories).Pictures were taken with a Leica microscope DM5000 B and Leica FireCam software.
Venus fluorescence.Dissected E8.5 embryos in ice cold PBS were analysed under a Leica microscope DMI6000 B using LAS AF software.
Skeletal preparations.Skeletons of E18.5 mouse foetuses and adult mice were dissected and stained with Alcian blue and Alizarin red following standard procedures [53].
Southern blot analysis.DNA was isolated from CHO attP cells, digested with various restriction enzymes (EcoRI, EcoRV, HindIII, MfeI, XbaI; NEB) over night and separated on a 0.7% agarose gel.Blotting, crosslinking, hybridisation (radioactively labelled DNA probe, i.e. a DraI/HindIII fragment of the pNZ-attP vector [57] containing the attP site and part of the Hyg r gene) and signal detection as for Northern blot analysis.
Biotinylation assay.Cells were plated on 6 cm dishes.At 80% confluence, the cells were washed twice with ice cold PBS supplemented with 0.1 mM CaCl 2 and 1 mM MgCl 2 (PBS-C/ M).After incubation on ice for 10 min with PBS-C/M, cells were treated with Sulfo-NHS-LC (Pierce; 0.25mg/ml in PBS-C/M) for 40 min to bind all proteins present on the cell surface.To quench the biotin reaction, cells were washed twice with PBS-C/M and incubated with 100 mM glycine in DMEM on ice for 30 min.After washing with PBS, the cells were lysed in lysis buffer [50 mM Tris-HCl pH7.6, 150 mM NaCl, 1 mM EDTA, 1% TritonX-100, 0.25% DOC (Sodium-desoxycholate), 0.1% SDS] supplemented with Complete Protease Inhibitor Cocktail Tablets (Roche) on ice for 30 min.Next, the samples were sonified and centrifuged at 10,000 x g for 15 min at 4°C to remove cell debris.Using NeutrAvidin beads (Thermo Scientific) prewashed in lysis buffer, biotinylated proteins were immunoprecipitated and analysed [95].Protein amounts were calculated as follows: total DLL protein in lysate = signal intensity of input x total volume of lysate/loaded volume; DLL protein on surface = signal intensity of IP x total volume of IP eluate/loaded volume; relative cell surface levels = DLL protein on surface / total DLL protein in lysate.
trans-activation assay.For analysis of Notch trans-activation, HeLa-N1 cells were transfected with 2 μg of RBP4-Luciferase reporter and 0.5 μg of firefly renilla-Luciferase on 6-well dishes using PerFectin (Genlantis).To analyse the effect of NOTCH1 glycosylation, HeLa-N1 cells were transiently transfected with 1.5 μg of attB-LfngHA.For analysis of cis-inhibition, HeLa-N1 cells were transiently transfected with 200 ng of Flag-tagged attB-Dll constructs.For analysis of the cis-inhibitory potential of NOTCH1, the ligand expressing CHO attP-DLL cells were transfected with 6 μg of Flag-tagged NOTCH1ΔC (NOTCH1 lacking the C-terminal 56 amino acids) expression vector.Expression of proteins was validated by Western blot analyses.Co-cultivation of HeLa-N1 and CHO attP cell lines was performed with 1.25 x10 5 cells each on 12-well dishes for 24 hours; pure HeLa-N1 cultures with 2.5 x10 5 cells on 12-well dishes for 48 hours.N-linked glycosylation was blocked by cultivating the cells in medium containing 1 μg/ ml tunicamycin for 22 hours.For Luciferase detection the Dual-Luciferase Reporter Assay System (Promega) was used according to the manufacturer's instructions; probes were analysed in a TurnerBioSystems luminometer with Glomax software.Each (co-)culture was performed in duplicates and every lysate was measured twice; the mean of the four measurements counted as n = 1.
Immunofluorescence.Cells were fixed in 4% formaldehyde for 10 minutes on ice, permeabilised with 1% Triton-X100 for 15 minutes at RT and washed in PBS for 30 minutes.Cells were then incubated for 5 minutes in 0.2% glycine, washed for 15 minutes with 0.1% Triton-X100 in PBS for 30 minutes, blocked with 5% FCS/0.1% Triton-X100/PBS for 1 hour at RT and incubated for 1 hour with primary antibodies (anti-Flag, clone M2, Sigma; 1:4,000), washed again with 0.1% Triton-X100 in PBS for 30 minutes and incubated with secondary antibodies (anti-mouse-Alexa488, Invitrogen; diluted in blocking solution 1:100) for 1 hour.After another washing step (0.1% Triton-X100 in PBS for 30 minutes) nuclei were stained by incubating the cells with TO-PRO3 (Invitrogen; diluted 1:1,000 in PBS) for 30 minutes at RT.After washing in PBS and water cells were mounted in ProLong-Gold antifade reagent (Life Technologies) and analysed with a Leica DM IRB microscope with a TCS SP2 AOBS scanhead.

Statistical analyses
Statistical analyses were performed using Prism software (GraphPad).Luciferase measurements were analysed by one-way ANOVA and activities obtained with each protein were compared using Bonferoni's Multiple Comparison Test with a significance level of 0.

Fig 1 .
Fig 1. 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; neo r , 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 Xchromosome 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.doi:10.1371/journal.pgen.1005328.g001 exon 11) common to Dll1 Dll1ki and Dll1 Dll4ki alleles (Fig 2Db, 2Dc and 2Df, black arrowheads).In situ hybridisation with a specific Dll1 ORF probe confirmed the absence of Dll1 transcripts in Dll1 Dll4ki homozygotes (Fig 2Dj, red arrowhead).Homozygous Dll1 Dll4ki embryos showed strong expression of Dll4 in the neural tube (Fig 2Dc, white arrow), reflecting activation of the Dll1 promoter in this region

Fig 2 .
Fig 2. Generation of Dll1 Dll4ki 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 neo r 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.neo r is removed by Cre-recombination.The resulting Dll1 Dll4ki allele and the Dll1 Dll1ki control are shown below (blue boxes, Dll1 mini gene).(B) Heterozygous adult Dll1 Dll4ki mice frequently (89%) displayed a kinky tail (arrow in b) but looked otherwise normal.(C) Heterozygous E15.5 Dll1 Dll4ki foetuses (c) were indistinguishable from wildtype (wt; a) and homozygous Dll1 Dll1ki (b) foetuses while all homozygous Dll1 Dll4ki foetuses (d) displayed shortened body axes and large oedemas.(D) Dll1 and Dll4 expression in Dll1 Dll4ki and Dll1 Dll1ki 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 Dll1 Dll4ki 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 Dll1 Dll4ki and Dll1 Dll1ki 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 Dll1 Dll4ki (a-f) and Dll1 Dll1ki 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 Dll1 Dll4ki (d) and of DLL4 signal in Dll1 Dll1ki 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.doi:10.1371/journal.pgen.1005328.g002

Fig 3 .
Fig 3. Homozygous Dll1 Dll4ki mice fail to generate proper somites and form reduced skeletal muscle tissue.Examination of Dll1-dependent (A,B) somitogenesis and (C-F) myogenesis in (a) wildtype, (b) Dll1 lacZ/lacZ , (c) Dll1 Dll1ki/Dll1ki , (d) Dll1 Dll4ki/+ and (e) Dll1 Dll4ki/Dll4ki embryos or foetuses.(A) Uncx4.1 in situ hybridisation of E9.5 embryos.(B) Skeletal preparations of E18.5 foetuses (Dll1 lacZ/lacZ foetuses do not survive until E18.5; red arrowheads indicate fused ribs or hemivertebrae in heterozygous Dll1 Dll4ki skeletons in d).(C) Myogenin in situ hybridisation to visualise differentiating skeletal muscle cells in myotomes of 17-18 somite stage embryos.(D, E,F) Anti-myosin heavy chain (MHC)-antibody staining of sectioned E15.5 foetuses showing intercostal muscles (D), the diaphragm (E), and muscles in the cross-section of forelimbs (F); black arrowheads indicate examples of muscle tissue, red arrowheads show lack of muscle tissue; asterisks label ribs (D) or bones of the forelimb (F).doi:10.1371/journal.pgen.1005328.g003 [54] as also observed in homozygous Dll1 lacZ embryos (Fig 3C, arrowheads;[37]).At E15.5, they had significantly less skeletal muscle tissue than wildtype or homozygous Dll1 Dll1ki foetuses but clearly more skeletal muscle tissue than Dll1 null mutants (Dll1 lacZ ) as shown for the intercostal muscles, the diaphragm and forelimbs by anti-MHC antibody staining of sectioned foetuses (Fig 3D-3F, arrowheads).These results indicate that DLL4 can partially substitute DLL1 during muscle cell differentiation and Dll1 Dll4ki behaves like a hypomorphic Dll1 allele.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 Dll1 Dll1ki/Dll1ki and Dll1 Dll4ki/Dll4ki retinas have a normal neuroepithelial organisation with a clear stratification of Chx10+ progenitors and p27+ differentiating neurons (Fig4B).Moreover, we find that similar numbers of early born retinal neurons [retinal ganglion cells (RGCs) and amacrine cells] are present in Dll1 Dll1ki and Dll1 Dll4ki retinas (Fig 4Cand 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 Dll1 Dll4ki/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 Dll1 Dll4ki/Dll4ki embryos.The extent of the functional equivalence of DLL1 and DLL4 depends on the developmental context.
We modified the co-culture assay by (transiently) expressing the ligands in the HeLa-N1 cells instead of in the CHO cells (Fig 5E, S7 Fig).In this setting, DLL ligands (expressed in HeLa-N1 cells) can trans-activate Notch in neighboring HeLa-N1+DLL cells (schematically shown in Fig 5Ec or in detail in S7A Fig); in addition, they can interact with Notch expressed in the same cell, i.e. in cis.When co-culturing HeLa-N1 cells expressing DLL1 with empty CHO cells (Fig 5Ea, S7 Fig), activation of Notch signalling was significantly increased as compared to a co-culture of HeLa-N1 cells expressing no transgenic DLL ligand with empty CHO cells

Fig 4 .
Fig 4. DLL4 expressed from the Dll1 locus rescues DLL1 loss-of-function in the retina.(A) Dll1 null 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 Dll1 Dll1ki and Dll1 Dll4ki 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 Dll1 Dll1ki and Dll1 Dll4ki 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 Dll1 Dll1ki (a,c) and in homozygous Dll1 Dll4ki (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 Dll1 Dll4ki/Dll4ki as compared to endogenous DLL4 expression in Dll1 Dll1ki/Dll1ki while signal strength is similar.Scale bars are 50 μm in (A, B) and 100 μm in (E).doi:10.1371/journal.pgen.1005328.g004

Fig 5 .
Fig 5. 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 CHO attP cells mediated by ΦC31 site-directed recombination (upper part).Resulting cells were used in Notchactivation 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 CHO attP-DLL1 (B5, C6) and CHO attP-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 CHO attP cells.(D) Notch trans-activation assays by co-culture of HeLa-N1 cells containing an RBP-Jκ:Luciferase reporter with CHO attP-DLL1 or CHO attP-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 CHO attP or CHO attP-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.doi:10.1371/journal.pgen.1005328.g005 Dll1 and Dll4 ORFs by swapping extracellular domains (resulting in DLL4-ICD+-TM/DLL1-ECD, termed DLL4-DLL1ECD, or DLL1-ICD+TM/DLL4-ECD, termed DLL1-DLL4ECD; ICD, intracellular domain; TM, transmembrane domain; ECD, extracellular domain; Fig 5G top).We introduced the chimeric Dll1-4 ORFs transiently into HeLa-N1 cells and performed co-culture assays analogous to the cis-inhibition experiments with non-chimeric DLL1 and DLL4 shown in Fig 5E (Fig 5G; S9 Fig).Measurement of Notch activity showed similarity between DLL1 and DLL4-DLL1ECD as well as between DLL4 and DLL1-DLL4ECD (Fig 5Ga', 5Gb' and 5Gc'; n = 6; compare with Fig 5Ea', 5Eb' and 5Ec').Particularly the statistically significant differences between bars in Fig 5Gb' clearly indicate that DLL4-DLL1ECD enhances, but DLL1-DLL4ECD reduces Notch activation by DLL1.As both chimeric ligands localise to the cell surface (S9B and S9C Fig; S6 Table) and are able to trans-activate Notch in a range similar to DLL1 and DLL4 (S9D Fig) these results

Fig 6 .
Fig 6.Model of Notch signalling in the PSM triggered by DLL1 and ectopic DLL4.Summary combining our in vivo and in vitro data in three different genetic scenarios (A-C); trans-activation (green arrows) and cisinhibition (red bars) in cells of the PSM are schematically depicted on the left, representative skeletal preparations to visualise the outcome of somitogenesis are shown on the right; references to Figs. in this paper are given below.(A) In wildtype and Dll1 Dll1ki/Dll1ki PSMs, endogenous or transgenic DLL1 (D1) transactivates Notch (N) signalling and results in a regularly segmented axial skeleton.(B) In our in vitro assays, DLL4 (D4) trans-activates Notch with similar efficiency as DLL1 but has an additional strong cis-inhibitory effect on Notch signalling that partially overrides trans-activation.The reduced net Notch activation in Dll1 Dll4ki/Dll4ki and CAG:Dll4;Dll1 loxP/loxP ;T(s):Cre PSMs is insufficient to support normal segmentation.(C) When both DLL1 and DLL4 are expressed (Dll1 Dll4ki/+ PSM), cis-inhibition by DLL4 plays a relatively smaller role, the resulting axial skeletons are mostly regular.However, cis-inhibition by DLL4 reduces the robustness of Notch signalling resulting in minor malformations (arrow indicates a misplaced rib), which are consistently seen in Dll1 Dll4ki/+ skeletons.doi:10.1371/journal.pgen.1005328.g006 CAG:Dll1 and CAG:Dll4 Hprt targeting constructs.Untagged Dll1 and Dll4 ORFs were PCR-amplified with primer pairs Dll1-for (SpeI) ACT AGT GCC ACC ATG TCT TAC GGT CAA GGG TCC AGC / Dll1-rev (AseI) GAT CAT TAA TTC ACA CCT CAG TCG CTA TAA CAC ACT CAT CCT TTT C and Dll4-for (NheI) GCT AGC AAT TCA TGA CGC CTG CGT CCC G / Dll4-rev (NdeI) CAT ATG TTA TAC CTC TGT GGC AAT CAC.Restriction sites introduced via primers were used to insert PCR products into NheI-NdeI sites of a shuttle vector containing IRES-GFP.Dll-IRES-GFP constructs were then subcloned into pMP8.CAG-Stop using restriction enzymes SwaI and MluI.HA-tagged versions of the above constructs for quantification of protein levels (Fig 1D) were cloned in a similar way: PCR primers for Dll1-HA were Dll1-for (SpeI; see above) / Dll1-HA-rev (AseI) ATT AAT CTA AGC GTA ATC TGG AAC ATC GTA TGG GTA CAT ACT AGA CAC CTC AGT CGC TAT AAC ACA C. A C-terminal HA-tag was introduced to the Dll4 ORF via gene synthesis (Life technologies) of the flanking regions of Dll4-HA lacking the central AflII/EcoRV Dll4 fragment that was cloned into the synthesised fragment; the complete Dll4-HA was cloned into NheI/NdeI of the shuttle vector.HA-tagged constructs were Cre-recombined in bacteria of the recombination strain SW106 (NCI at Frederick).Dll4ki targeting construct.The Dll1 mini gene in the Dll1 Dll1ki (Dll1 tm2Gos ; Fig 2A bottom; [37]) targeting vector was replaced with a Dll4 mini gene by a 3-point ligation of a NsiI/KpnI fragment of the Dll1ki targeting construct (containing DT, the 5' homology region and the 5'UTR of Dll1 exon1), a NsiI/SmaI fragment of the same Dll1ki targeting vector (containing the vector backbone with amp r , 3' homology region and floxed PGK-neo r ) and a KpnI/PmeI fragment (containing the complete Dll4 ORF with start codon ATG followed by 3 Stop codons and-as an inactive remainder of a precursor clone-the genomic Dll1 region from the last 42 bp of exon 9 to exon 11; gene structure resembled a Dll3ki targeting vector, [21]).To adjust the gene structure of the Dll4ki targeting vector to that of control Dll1ki, a 1,466 bp MfeI/BsiWI fragment (comprising last 134 bp of Dll4 exon 9, the genomic Dll1 remainder from exon 9-11 and 154 bp downstream) was replaced with a synthesised 1,293 bp MfeI/BsiWI DNA fragment (comprising Dll4 exon 9 from the MfeI site, Dll1 intron 9, Dll4 exon 10, Dll1 intron 10, Dll1 exon 11 and 154 bp downstream; Life technologies); the only remaining difference between Dll4ki and control Dll1ki targeting vectors were the coding regions of Dll4 or Dll1 mini gene (ORFs from exon 1 to exon 9 and separate exon 10; exon 11 contains only one coding amino acid, i.e. a conserved valine).
by AfeI/HindIII digest, blunting ends with T4 DNA-Polymerase (Roche) and religation; Flag-tagged Dll1 and Dll4 ORFs were released from pTracer-CMV and inserted into pNC-attB-deltaGFP using restriction enzymes EcoRI/BamHI.To generate an alternative construct with HA-tagged DLL4, the HA-tag was added to the Dll4 ORF by PCR with primer pair Dll4.up (EcoRI) GAA TTC ACC ATG ACG CCT GCG TCC CGG AGC G / Dll4.lowHA (NotI) GCG GCC GCT TAT TAT TAA GCG TAG TCT GGA ACG TCG TAT GGG TAT ACC TCT GTG GCA ATC ACA CAC TCG.Dll4-HA was inserted into EcoRI/NotI sites of pTracer-CMV and subcloned with PmeI/XbaI into AfeI/XbaI sites of pNC-attB-deltaGFP.Chimeric Dll1-4 constructs were partly generated by gene syntheses (Life technologies) and cloned into pNC-attBdeltaGFP.For Dll4-Dll1ECD an NdeI-EcoRV fragment containing a part of Dll1 ECD and complete Dll4 TM and ICD synthesised; a BglII/NdeI Dll1 ECD fragment was inserted into the gene synthesis vector and the whole chimeric ORF was inserted into pNC-attBdeltaGFP-Dll4 as an EcoRI/EcoRV fragment.For Dll1-Dll4ECD a BspEI/MfeI fragment containing part of Dll4 ECD, Dll1 ICD and part of Dll1 ICD was synthesised.Dll1 ICD was inserted as a MfeI/XbaI fragment and Dll4 ECD as a ScaI/BspEI fragment; the chimeric ORF was then inserted into pNC-attBdeltaGFP-Dll4 as a ScaI/XbaI fragment.pNC-attBdeltaGFP with chimeric Dll1-4 were used as transiently transfected expression vectors (CMV promoter).
05. Means for all three DLL1-and DLL4-HA clones in Fig 1D, cell counts in the retina and cell surface levels of chimeric ligands were analysed using the Student's t-test.S3 Fig. Defects in the axial skeletons of heterozygous Dll1 Dll4ki/+ adults.Skeletal preparations of seven adult Dll1 Dll4ki/+ males (1-7; 4 to 8 months old) are largely normal but consistently exhibit irregularities (arrows) in the rib cage (top) and/or tail (bottom) suggesting a mild dominant-negative effect of transgenic Dll4 (see main text and Discussion).(TIF) S4 Fig. Validation of unique attP site integration in CHO attP cells.(A) Map of the genomic integration of the pHZ-attP construct containing attP site, Hyg r , Zeo r and frt-flanked Jagged1 (Jag1).The position of restriction sites and of the probe used for Southern blot analysis are indicated.The flanking genomic sequence and position of restriction sites outside the vector is unknown.(B) Southern blot analysis of DNA isolated from CHO attP-JAG1 cells shows a single product for each digest indicating a single genomic integration of the attP construct (the 3.3 kb EcoRI fragment is entirely derived from the integrated construct and served as a control).(C) FC31 integrase-mediated insertion of Dll1 and Dll4 into CHO attP-JAG1 generates CHO attP-- JAG1-DLL1 or CHO attP-JAG1-DLL4 cells used in S6D Fig; JAG1 is Myc-tagged, DLL1 and DLL4 are Flag-tagged.(D) Excision of Jag1 by FLP recombination results in CHO attP cells that were subsequently used for the generation of CHO attP-DLL1 and CHO attP-DLL4 cells.(TIF) S5 Fig. DLL1 and DLL4 stably expressed in CHO attP cells: Protein levels, surface localisation and half-lives.(A) Exemplary Western blot used for the analysis of protein levels in Fig 5B.CHO attP cells were used as negative control; β-actin was used for normalisation.(B) Extended Western blot analysis of protein levels including additional clones of CHO attP-DLL1 and CHO attP-DLL4 .Expression levels varied to some degree, but DLL4 levels were not below DLL1 levels.Clone CHO attP-DLL1 C6 is the same in Fig 5B and can be used to compare values between both Figs.Error bars represent SEM; ns, not significant; Ã , P<0.05; ÃÃ , P<0.01.(C) Exemplary Western blot used for the quantification of cell surface protein levels by biotinylation in Fig 5C.The protein amount was quantitated and the relative protein surface level was calculated as described in Materials and Methods.(D) Immunocytochemistry of fixed CHO attP-DLL1 and CHO attP-DLL4 cells.Flag-tagged ligands were visualised using anti-Flag antibodies.DLL1 and DLL4 are present at the cell surface.(E,F) Determination of DLL1 and DLL4 protein half-lives.(E) DLL1 and DLL4 half-lives analysed using two different clones for each cell line.(F) Average protein decay of the clones shown in (E): DLL4 is more stable (half-life 7.3 hours) than DLL1 (half-life 4.9 hours).Dashed lines indicate the 95% confidence interval.(TIF) S6 Fig. Influence of the presence of LFNG or JAG1 or of inhibition of N-glycosylation on Notch activation.(A) Notch trans-activation assays with co-cultures of CHO attP (negative control), CHO attP-DLL1 and CHO attP-DLL4 cells with Notch reporter expressing HeLa-N1 cells (cf.Fig 5D) without and with transient expression of LFNG-HA.Expression of LFNG in HeLa-N1 cells decreases the trans-activation ability of DLL4 to levels similar to DLL1, whose activation potential is slightly increased.(B) Scheme of interactions in co-cultivation assays with possible influence of LFNG expressed in HeLa-N1 cells.(C) Western blot showing the expression of LFNG-HA in HeLa-N1 cells used in (A); β-tubulin, loading control.(D) Notch trans-activation assays with CHO attP , CHO attP-DLL1-Flag and CHO attP-DLL4-HA cells without or with stable expression of JAG1-Myc (S4C and S4D Fig) in co-culture with Notch reporter expressing HeLa-N1 cells.Stable coexpression of JAG1 in DLL1 or DLL4 presenting cells does not significantly change Notch activation.DLL4 plus JAG1 activate the receptor more efficiently than DLL1 plus JAG1.(E) Scheme of different possible interactions in co-cultivation assays with or without stable JAG1 expression.(F) Western blot showing the expression of DLL1-Flag, 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).