The ectodomains determine ligand function in vivo and selectivity of DLL1 and DLL4 toward NOTCH1 and NOTCH2 in vitro
The Notch signaling pathway mediates communication between neighboring cells in metazoans and thereby regulates a multitude of developmental processes in various tissues ( Artavanis-Tsakonas et al . , 1995; Yoon and Gaiano , 2005; Bolós et al . , 2007; Gridley , 2007; Radtke et al . , 2010; Koch and Radtke , 2011; reviewed in Louvi and Artavanis-Tsakonas , 2012; Kopan , 2012 ) . This communication depends on the interaction of Notch receptors on the surface of the signal receiving cells with transmembrane ligands on the surface of adjacent cells . Ligand binding then leads to a sequence of proteolytic cleavages of the receptor releasing the Notch intracellular domain ( NICD ) from the membrane . NICD translocates into the nucleus where it enters into a complex with a CSL protein ( CBF-1/RBPJ in mammals , Suppressor of Hairless in flies , and Lag-1 in worms ) and a protein of the Mastermind family ( Petcherski and Kimble , 2000; Wu et al . , 2000; Nam et al . , 2003; Nam et al . , 2006; Wilson and Kovall , 2006; Choi et al . , 2012 ) to regulate transcription of target genes ( reviewed in Bray , 2016 ) . Mammals have four Notch receptors ( N1-N4 ) and four activating ligands of the DSL ( Delta , Serrate , LAG-2 ) family: DLL1 and DLL4 , orthologs of Drosophila Delta , and JAG1 and JAG2 , orthologs of Drosophila Serrate . DLL1 and DLL4 are similar in domain structure , size and sequence ( Shutter et al . , 2000 ) . Both proteins contain an N-terminal MNNL ( also referred to as C2 ) domain ( Chillakuri et al . , 2013; Suckling et al . , 2017 ) , followed by a DSL domain and eight EGF-like repeats in their extracellular portion , and a less well conserved intracellular domain . The MNNL and DSL domains , required for high-affinity binding of Delta-like ligands to Notch receptors ( Rebay et al . , 1991; Cordle et al . , 2008 ) , contact EGF repeats 12 and 11 of Notch , respectively ( Luca et al . , 2015 ) . Contributions from adjacent EGF-like repeats , however , are required for signal transduction by Delta-like ligands ( Andrawes et al . , 2013 ) as well as for optimal interaction with Serrate ( Yamamoto et al . , 2012 ) and Jagged ( JAG ) -family ligands ( Luca et al . , 2017 ) . Although the biological activities of DLL1 and DLL4 are partially overlapping , the two proteins are not equivalent in vitro or in vivo . In cell culture studies , DLL4 is more effective than DLL1 in activating N1 signaling during T cell development ( Besseyrias et al . , 2007 ) , consistent with its ten-fold higher binding affinity in binding studies using purified fragments of N1 , DLL1 , and DLL4 ( Andrawes et al . , 2013 ) . In vivo , studies of adult intestinal epithelium in mice have shown that DLL1 and DLL4 are co-expressed in crypts and act redundantly to maintain the intestinal stem cell pool ( Pellegrinet et al . , 2011 ) . In contrast , however , mouse DLL1 cannot fully replace DLL4 in its ability to trigger T lineage commitment ( Besseyrias et al . , 2007; Mohtashami et al . , 2010 ) . Conversely , endogenous DLL4 does not substitute for DLL1 in its ability to promote development of the arterial vascular epithelium ( Sörensen et al . , 2009 ) , nor does it compensate for the function of DLL1 in the paraxial mesoderm , a tissue where these ligands are normally not co-expressed: mice in which DLL1 was replaced by DLL4 had severe somite patterning defects and showed premature myogenic differentiation leading to reduced skeletal muscles . However , the function of DLL1 during early retina development was rescued by DLL4 in these mice ( Preuße et al . , 2015 ) . Collectively these studies indicate that the functionality of DLL1 and DLL4 strongly depends on context , but it remains unclear which portions of these similar DSL proteins account for their functional non-equivalence . A recent study in cell culture observed that DLL1 and DLL4 stimulate NOTCH1 receptors to produce responses with different dynamics , attributing differences between pulsatile signaling of DLL1 and sustained signaling by DLL4 to the intracellular , rather than the extracellular , regions of the proteins ( Nandagopal et al . , 2018 ) . Here , we investigate the influence of the extracellular and intracellular regions of DLL1 and DLL4 chimeric proteins on ligand function in cell culture assays , and for selected chimeras , in biochemical binding assays and in vivo in mice . We observe that in vivo differences of DLL1 and DLL4 function during somite patterning and myogenesis are encoded by the ligands ectodomains , that DLL1 and DLL4 are able to discriminate between NOTCH1 and NOTCH2 in vitro , and that ligand residues outside of the known binding interface are important contributing factors for ligand function in vivo .
Previous in vivo analyses indicated that DLL4 cannot substitute for DLL1 function during embryonic development ( Preuße et al . , 2015 ) . To test whether the inability of DLL4 to rescue the loss of DLL1 in the paraxial mesoderm in vivo resides in its extra- or intracellular domain we generated single copy transgenic mice allowing for the conditional expression of chimeric DLL molecules consisting of the extracellular domain of one ligand and transmembrane and intracellular domain of the other ( D1ECD_D4ICD and D4ECD_D1ICD , II and VII in Figure 1 ) . Transgenes were introduced into the Hprt locus of Hprt-deficient E14TG2a ES cells by homologous recombination using the strategy already employed for the initial analysis of full length DLL1 and DLL4 during somitogenesis ( Preuße et al . , 2015 ) . Briefly , cDNAs encoding chimeric ligands were cloned into the targeting vector pMP8 in reverse orientation downstream of neomycin phosphotransferase ( neor ) driven by the CAG promoter . Cre-mediated recombination of two loxP sites and two mutant loxP2272 ( loxM ) sites removes the neor cassette and flips the gene of interest and results in its expression from the CAG promoter ( Figure 2A ) . To test whether the extracellular or intracellular domain determines the inability of DLL4 to rescue the loss of DLL1 in mesodermal tissues of early embryos , we induced expression of either chimeric ligand and simultaneously removed endogenous DLL1 using a floxed Dll1 allele and a Cre transgene expressed in the primitive streak driven by a promoter derived from brachyury ( T ( s ) :Cre ) ( Feller et al . , 2008 ) . Because the Hprt locus is located on the X-chromosome , we used hemizygous male embryos for the analysis . As previously described , inactivation of Dll1 in the mesoderm resulted in loss of Uncx ( formerly called Uncx4 . 1 ) expression in caudal somite compartments ( n = 12; Figure 2Bb , b' ) indicating severe somite patterning defects compared to wild type embryos ( n = 28; Figure 2Ba , a' ) . Expression of D1ECD_D4ICD in Dll1-deficient embryos ( n = 9 ) restored robust expression of Uncx similar to full length DLL1 ( Preuße et al . , 2015 ) . Uncx expression expanded into cranial somite compartments ( Figure 2Bc , c' ) reminiscent of ectopic Notch activity ( Feller et al . , 2008 ) , probably reflecting non-restricted D1ECD_D4ICD expression throughout the PSM and somites . In contrast , expression of D4ECD_D1ICD barely restored Uncx expression in the majority ( n = 8/12 ) of Dll1-deficient embryos ( Figure 2Bd , d' ) , a phenotype similar to that seen with full-length DLL4 ( Preuße et al . , 2015 ) , even though the chimeric ligand was expressed and detected on the cell surface of PSM cells ( Figure 2Cd-f ) . As observed previously for full-length DLL4 ( Preuße et al . , 2015 ) some embryos ( n = 4 ) displayed essentially normal Uncx expression ( not shown ) , which might result from some perdurance of DLL1 activity or delayed or inefficient excision of endogenous Dll1 . Overall , this analysis strongly suggests that the functional difference between DLL1 and DLL4 observed in vivo during somitogenesis resides in the extracellular domains . The N-terminal MNNL and DSL domains and adjacent EGF repeats 1–3 constitute the major interface for interaction between DSL ligands and Notch receptors , and are essential for ( full ) activation of Notch signaling ( Cordle et al . , 2008; Andrawes et al . , 2013; Luca et al . , 2015; Schuster-Gossler et al . , 2016; Luca et al . , 2017 ) . To analyze whether this region accounts for the observed differences between DLL1 and DLL4 in vivo we generated a chimeric ligand that contained the N-terminal region up to and including EGF3 of DLL1 fused to EGF4 and the remaining C-terminal portion of DLL4 ( D1N-E3_D4 , III in Figure 1; the amino acid sequence around the fusion is shown in Figure 1—figure supplement 1Ab ) . We then tested whether this chimeric ligand is sufficient for normal DLL1 function during development . We generated mice ( Dll1D1N-E3_D4ki ) expressing D1N-E3_D4 instead of DLL1 using the "mini-gene“ knock-in strategy ( Figure 3A ) that disrupts endogenous Dll1 , successfully employed previously to express either a Dll4 or Dll1 ( control ) mini-gene ( Schuster-Gossler et al . , 2007; Preuße et al . , 2015; Schuster-Gossler et al . , 2016 ) . Heterozygous mice obtained from two independent targeting events carrying the Dll1D1N-E3_D4ki allele were viable and showed no apparent phenotype . Homozygous Dll1D1N-E3_D4ki mice were stillborn ( n = 3 and 4 , respectively ) , indicating that D1N-E3_D4 cannot fully replace DLL1 during development although it is present on the cell surface of PSM cells ( Figure 3Cd-f ) . At E15 . 5 homozygous Dll1D1N-E3_D4ki fetuses showed a stumpy tail ( n = 5 and 6 , respectively; arrow in Figure 3Bb ) similar to Dll1Dll4ki mutants ( Figure 3Bd ) ; however , they lacked the edema observed in Dll1Dll4ki homozygotes ( arrowhead in Figure 3Bd ) . D1N-E3_D4 was also not able to restore normal Uncx expression ( Figure 3De ) . Axial skeletons of homozygous Dll1D1N-E3_D4ki fetuses were severely disorganized ( n = 10; Figure 3Ef ) , a phenotype consistent with abnormal Uncx expression and similar to Dll1Dll4ki/Dll4ki homozygote axial skeletons ( Figure 3Ed ) , although the rib cage appeared less compressed . In contrast to Dll1Dll4ki heterozygotes ( Figure 3Ec; Preuße et al . , 2015 ) , which often displayed axial skeleton defects ( n = 14/16 ) such as hemivertebrae ( arrow in Figure 3Ec ) and fused ribs ( arrowheads in Figure 3Ec ) heterozygous Dll1D1N-E3_D4ki fetuses showed no defects of the axial skeleton ( n = 0/14; Figure 3Ee ) indicating that D1N-E3_D4 lacks the dominant interfering activity of DLL4 . Deletion of DLL1 during myogenesis leads to premature differentiation of myogenic progenitor cells resulting in severe skeletal muscle hypotrophy at fetal stages ( Schuster-Gossler et al . , 2007 ) . This phenotype cannot be suppressed by DLL4 expression ( Figure 3Fc; Preuße et al . , 2015 ) . In contrast , skeletal muscles of Dll1D1N-E3_D4ki/D1N-E3_D4ki homozygotes ( Figure 3Fd-f; n = 3 ) were virtually indistinguishable from Dll1Dll1ki/Dll1ki ( Figure 3Fb ) and wild type fetuses ( Figure 3Fa ) . These in vivo analyses indicate that , unlike the D1ECD_D4ICD chimera , D1N-E3_D4 is not a fully functional DLL1 ligand during somite patterning . However , D1N-E3_D4 remains functional during myogenesis and restricts muscle progenitor differentiation despite the presence of the DLL4 ICD , consistent with the conclusion that in vivo the functional difference between DLL1 and DLL4 is encoded in the ECDs . In cell-based trans-activation assays using HeLa cells stably expressing murine N1 ( HeLaN1 ) co-cultured with CHO cells expressing mouse DLL1 ( mDLL1 ) or DLL4 from the same locus both ligands activated a transiently expressed Notch reporter similarly ( Preuße et al . , 2015 ) . However , a purified fragment of the extracellular domain of human DLL4 ( N-terminus up to and including EGF5: hD4N-E5 ) bound to hN1 with an approximately ten-fold higher affinity than the corresponding hDLL1 fragment ( Andrawes et al . , 2013 ) . Like hD4N-E5 , mD4N-E5 has a higher affinity for hN1 ( KD = 0 . 43 ± 0 . 046 µM; Figure 1—figure supplement 2Aa ) than the corresponding mDLL1 fragment ( KD = 1 . 56 ± 0 . 207 µM; Figure 1—figure supplement 2Ab ) , as judged by biolayer interferometry measurements . To find a potential explanation for the discrepancy between binding affinities and Notch activation in HeLaN1 cells we analyzed these cells for expression of other Notch receptors and found that in addition to exogenous mouse Notch1 HeLaN1 cells express endogenous NOTCH2 and NOTCH3 ( Figure 4A ) , which might have masked underlying differences in the intrinsic N1 response to the DLL1 and DLL4 ligands . To detect potential differences in ligand activity towards N1 or N2 , the two Notch receptors present during somitogenesis , and to reduce variability due to transient reporter expression we stably integrated a Notch luciferase reporter in the Hprt locus ( E14rep ) of mouse E14TG2a ES ( E14 ) cells , and generated stable cell lines expressing either Notch1 ( N1rep ) or Notch2 ( N2rep ) in these cells ( Schuster-Gossler et al . , 2016 ) . When co-cultured with E14 cells or DLL1 or DLL4 expressing cells , E14rep cells show luciferase activity similar to wild type E14 levels ( compare I to II , III , and IV in Figure 4B; numerical values in Figure 4—source data 1 ) , indicating that negligible amounts of functional endogenous NOTCH receptors are present in E14 cells . Likewise , N1rep cells show essentially no activation above background when co-cultured with wild type E14 ES cells ( compare V to I in Figure 4B; numerical values in Figure 4—source data 1 ) , indicating insignificant amounts of functional endogenous Notch ligands in these cells . ES cells expressing exogenous DLL1 activate the luciferase reporter approximately ten-fold above the basal signal in E14 ES cells when co-cultured with N1rep cells ( compare VI to V in Figure 4B; numerical values in Figure 4—source data 1 ) indicating that our co-culture system reliably measures specific Notch signaling activity . To create ligand presenting cells for a comparison between mDLL1 and mDLL4 , we generated ES cells expressing either mDLL1 or mDLL4 from single copy integrations into the Hprt locus . Co-cultures ( n = 39 ) of cells expressing DLL1 or DLL4 with N1rep ES cells consistently revealed higher activation of N1 by DLL4 than by DLL1 ( mean 12 . 454 ± 3 . 961 SD fold of non-normalized luciferase activity , 9 . 42 ± 2 . 997 SD fold , when normalized to DLL1 activation and corrected for protein expression and cell surface levels ( Figure 4C–E; numerical values Figure 4—source data 2 , Figure 4—source data 3 , Figure 4—source data 4 ) . In contrast , DLL4 activated N2 significantly less efficiently than did DLL1 ( n = 39; mean 0 . 468 ± 0 . 161 SD fold of non-normalized luciferase activity , 0 . 35 ± 0 . 12 SD fold , when normalized to DLL1 activation and corrected for protein expression and cell surface levels ( Figure 4F; numerical values in Figure 4—source data 5 ) . To confirm that the observed differences between DLL1 and DLL4 in activating N1 and N2 were not a secondary consequence of clonal selection ( however unlikely ) , we also analyzed additional DLL1 ( n = 3 ) and DLL4 ( n = 9 ) expressing ES cell clones for protein expression and N1 or N2 activation . Despite some variability of protein expression ( Figure 4—figure supplement 1B and Figure 4—figure supplement 1—source data 1 ) and Notch activation levels between individual clones and co-cultures , all DLL4 clones consistently activated N1 significantly better than all DLL1 clones , and all DLL4 clones stimulated N2 significantly less efficiently than DLL1 ( Figure 4—figure supplement 1C , D; numerical values in Figure 4—figure supplement 1—Source Data 2 ) , indicating that both ligands differ significantly in their ability to activate different Notch receptors in our cell-based assay . Consistent with the differences in N2 stimulation by mDLL1- and mDLL4-expressing cells , the highly homologous human hD1N-E5 exhibits a higher affinity ( KD = 0 . 36 ± 0 . 11 µM; Figure 1—figure supplement 2Ba ) for human NOTCH2 than D4N-E5 ( KD = 1 . 28 ± 0 . 2 µM; Figure 1—figure supplement 2Bb ) . In an attempt to identify the domains of DLL1 and DLL4 that contribute to differences in activating N1 and N2 , we carried out a series of domain swaps to generate a set of chimeric ligands ( II-V , VII-X in Figure 1 ) for stimulation of N1 and N2-expressing cells in our co-culture assay . Like wild-type ligands , chimeric ligands were expressed from single copy transgene integrations in the Hprt locus of murine ES cells . All chimeric proteins were expressed and present on the cell surface ( Supplementary file 1 ) , but expression levels varied among the chimeras ( Supplementary file 2 ) despite integration into the Hprt locus by homologous recombination . We thus analyzed receptor selectivity of the chimeras in stimulating N1 and N2 responses using the co-culture assay by determining the N1/N2 response ratio . Stimulation with DLL1 gives a N1/N2 response ratio of approximately 1 , DLL4 of ~20 ( Figure 5A; numerical values used for calculations in Figure 5—source data 1 , Figure 5—source data 2 , Figure 5—source data 3; graphical representations of the relative luciferase activities of the ligands are shown in Figure 5—figure supplement 1 ) . Strikingly , chimeras which retain the full ectodomain or the MNNL-EGF3 region of DLL4 have a N1/N2 stimulation ratio of approximately 20 similar to DLL4 , whereas chimeras that retain the ectodomain , or MNNL-EGF3 region of DLL1 have a stimulation ratio of between one and two , resembling DLL1 ( Figure 5A ) . These results indicate that the differences in activation potential of DLL4 and DLL1 toward N1 and N2 are encoded in the N-terminal part of the protein , encompassed by MNNL-EGF3 . When chimeras include the MNNL-EGF2 or MNNL-DSL region of one ligand and the remainder of the other , the N1/N2 stimulation ratios of the chimeric pairs are equivalent ( Figure 5A ) , indicating that the third EGF-like repeat makes an important contribution to receptor selectivity . To analyze to what extent the amino acids that contact N1 in the binding interfaces of the MNNL and DSL domains might contribute to the different activity of DLL1 and DLL4 toward N1 and N2 we reciprocally exchanged these amino acids ( XI-XII in Figure 1; Figure 5C and Figure 1—figure supplement 1B ) based on alignments of the DLL4 ( Luca et al . , 2015 ) and DLL1 ( Kershaw et al . , 2015 ) structures ( Figure 5B ) . Western blot analyses of cell lysates and cell surface biotinylation and immunoprecipitation showed that all variants were present on the cell surface ( Supplementary File 1 ) . The N1/N2 response ratios show that swapping the contact residues of DLL4 onto DLL1 do not substantially affect the activation ratio when compared to DLL1 itself , indicating that the differences between DLL1 and DLL4 in N1/N2 selectivity cannot simply be accounted for by interfacial residues in the MNNL-DSL region ( Figure 5D; numerical values used for calculations are in Figure 5—source data 1 , Figure 5—source data 2 , Figure 5—source data 3 ) . Similarly , replacement of the DLL4 contact residues by the analogous residues of DLL1 slightly reduces the mean N1/N2 activation ratio ( to ~13 ) , but does not collapse the ratio to 1 ( Figure 5D ) , again strongly suggesting that residues outside of the MNNL-DSL contact interface contribute to the relative N1 selectivity of DLL4 . These results are consistent with 1 ) the domain swap data , which argue that discrimination between DLL1 and DLL4 depends on the EGF repeats as well as on the MNNL-DSL region , and 2 ) the prior observation that variants of DLL4 selected for high N1 affinity accumulate mutations in the protein core , but not in the binding interface ( Luca et al . , 2015 ) . Swapping the contact residues of DLL1 onto DLL4 did not reduce the binding affinity of DLL4 for N1 ( D4contD1 KD = 0 . 327 ± 0 . 036 µM; Figure 1—figure supplement 2Ac ) , fully consistent with the interpretation that the protein core of DLL4 contributes to N1 binding affinity , likely by influencing the fraction of molecules in a binding-active conformation . Although swapping the contact residues of DLL4 onto DLL1 increased binding affinity for N1 ( D1contD4 KD = 0 . 326 ± 0 . 044 µM; Figure 1—figure supplement 2Ad ) , the substitution did not substantially change the N1/N2 activation ratio , indicating that binding affinity for N1 is not the only influence on the selectivity of the two ligands for N1 or N2 . The DLL4 MNNL domain contains three N-glycosylation sites , one of which ( N109 ) is conserved from amphibian to mammalian DLL4 ligands but absent in DLL1 . This residue resides adjacent to the contact amino acid F110 ( Figure 1—figure supplement 3A ) . We confirmed that DLL4 can actually be N-glycosylated at this site ( Figure 1—figure supplement 3C ) and tested whether N109-glycosylation contributes to DLL4 activity and selectivity by mutating N109 to G ( XIII in Figure 1 ) , which is the amino acid present in DLL1 in the equivalent position ( G112 ) . D4N109G had no effect on the relative activation potential of DLL4 for N1 versus N2 ( Figure 5D ) , and its affinity for N1 was not altered ( KD = 0 . 341 ± 0 . 015 µM; Figure 1—figure supplement 2Ae ) , indicating that N-glycosylation at this site does not significantly modulate N1 binding or contribute to the relative selectivity of DLL4 towards N1 and N2 . To test whether the contact amino acids of DLL1 and DLL4 contribute to their functional divergence in vivo we generated a mouse line expressing D1contD4 ( XI in Figure 1 ) instead of wild type DLL1 using our "mini-gene" knock-in strategy ( Figure 3A ) . Heterozygous mice carrying this allele ( Dll1D1contD4ki ) are indistinguishable from wild type . Homozygous mutants obtained from heterozygous matings at the expected Mendelian ratio ( 6/27 ) were viable and fertile , and indistinguishable from wild type and Dll1Dll1ki/Dll1ki controls ( Figure 6A ) . Uncx was expressed in regular pattern in the caudal halves of the somites of homozygous embryos ( Figure 6Cd , d’ ) , consistent with only subtle abnormalities of individual vertebral bodies in the lower thoracic region of Dll1D1contD4ki/D1contD4ki fetuses ( Figure 6D; n = 3/4 ) indicating that the contact amino acids and different binding affinities are not a major discriminating feature of the two ligands in vivo .
DLL1 and DLL4 have context-dependent redundant and divergent functions , but the bases for these differences are unclear . Here , using systematic domain exchanges and mutation of contact amino acids in the MNNL and DSL domains of DLL1 and DLL4 , cell-based and biochemical assays , and transgenic mice we show that ( 1 ) DLL1 and DLL4 differ significantly in their potential to activate N1 and N2 and this difference is encoded in the ligand ectodomains , ( 2 ) regions outside the known contact interface contribute to context-dependent ligand function , and ( 3 ) the contact amino acids are not the sole or primary determinant of this discrimination between the two receptors . Analysis of our transgenic mice expressing D1ECD_D4ICD or D4ECD_D1ICD indicate a critical role of the ECD for the function of DLL1 during somite patterning in vivo . This resembles intrinsic functional differences that reside in the extracellular domains of mN1 and mN2 during kidney development ( Liu et al . , 2013 ) , whereas the N1 and N2 ICDs appear to be functionally equivalent in various developmental contexts ( Liu et al . , 2015 ) . Functional equivalence of DLL1’s and DLL4’s ICDs in vivo is further supported by the rescue of the skeletal muscle phenotype in our D1N-E3_D4 knock-in mice , which harbor the DLL4 ICD . In this developmental context even domains C-terminal to EGF3 of DLL1 appear to be interchangeable . Analyses of the ECD/ICD domain swaps in the cell-based assay also suggest that the discriminatory potential of the ligands tracks with the ECD , and not with the ICD , even though the ICD appears to affect the strength and/or dynamics of the signal in co-culture assays where ligand and receptor expression is enforced in vitro ( Nandagopal et al . , 2018 ) . Additional sources of complexity in vivo , like the stronger cis-inhibitory potential of the DLL4 ECD on Notch signaling ( Preuße et al . , 2015 ) , or cyclic modulation of Notch by LFNG in the paraxial mesoderm , or different interactions with lipids ( Suckling et al . , 2017 ) might account for the resistance to loss of function phenotypes from ligand ICD swaps in vivo . EGF-like repeats 11 and 12 of mouse N1 and N2 are highly similar ( 56/83 residues identical , 14 similar amino acids ) , and 13 of the 17 amino acid residues at the DLL4-binding interface are identical . Moreover , the x-ray structures of the EGF11-13 fragments of human N1 and N2 adopt a very similar arrangement ( Suckling et al . , 2017 ) . Nevertheless , DLL1 and DLL4 exhibit a "discrimination potential" of ~20 fold in terms of receptor response in culture assays , suggesting that either the few different contact amino acids in EGF 11 and 12 of N1 and N2 have a significant impact or interactions of DLL1 and DLL4 with N1 and N2 are not limited to the MNNL and DSL interfaces with receptor EGF repeats 11 and 12 . Domain swaps carried out here show that the region responsible for this receptor discrimination maps to the MNNL-EGF3 region ( Figure 5 ) . These findings are consistent with previous work uncovering the requirement of EGF repeats 1–3 of the DLL ligands for NOTCH1 activation , the importance of this region in the binding of Serrate family ligands to Notch receptors and in Serrate/Jagged-induced signaling , and the importance of EGF repeats 8–10 of NOTCH1 for signal activation by DLL ligands ( Shimizu et al . , 1999; Cordle et al . , 2008; Yamamoto et al . , 2012; Andrawes et al . , 2013; Schuster-Gossler et al . , 2016; Luca et al . , 2017; Liu et al . , 2017 ) . Together , this body of work suggests that interactions of the N-terminal EGF repeats of the DLL ligands with EGF repeats 8–10 of Notch also contribute to recognition and impart discriminatory potential . The D1N-E3_D4 knock-in mice also point a functional role for domains outside the known binding interface , since this chimeric ligand does not substitute fully for DLL1 in vivo during somite patterning despite harboring the MNNL and DSL domains and EGF1-3 of DLL1 , supporting context-dependent contributions of additional C-terminal EGF repeats observed previously in mice ( Schuster-Gossler et al . , 2016 ) . Remarkably , the exchange of the contact amino acids in DLL1 with those of DLL4 in the D1contD4 protein does not alter receptor selectivity in cultured cells even though these changes increase N1 binding affinity . This result suggests that receptor selectivity of DLL1 and DLL4 is not determined exclusively by the differences in binding strength . The D1contD4 chimera even substitutes almost completely for DLL1 function in mice during somite patterning , which is highly sensitive to altered Notch signaling ( Schuster-Gossler et al . , 2009 ) and therefore a suitable in vivo read out to detect even minor differences of Notch ligand function . Together , our results also favor the conclusion that the contact amino acids in the MNNL and DSL domains do not make the dominant contributions to the functional divergence of DLL1 and DLL4 in vivo , suggesting instead that differences in the domain cores , and/or contacts outside of the known DLL4-NOTCH1 interface , are the factors that most contribute to this functional divergence .
DLL1 and DLL4 are Notch ligands with high structural similarity but context-dependent functional differences . Here , we analyze their functional divergence using cellular co-culture assays , biochemical studies , and in vivo experiments . DLL1 and DLL4 activate NOTCH1 and NOTCH2 differently in cell-based assays and this discriminating potential lies in the region between the N-terminus and EGF repeat three . Mice expressing chimeric ligands indicate that the ectodomains dictate ligand function during somitogenesis , and that during myogenesis even regions C-terminal to EGF3 are interchangeable . Substitution of NOTCH1-interface residues in the MNNL and DSL domains of DLL1 with the corresponding amino acids of DLL4 , however , does not disrupt DLL1 function in vivo . Collectively , our data show that DLL4 preferentially activates NOTCH1 over NOTCH2 , whereas DLL1 is equally effective in activating NOTCH1 and NOTCH2 , establishing that the ectodomains dictate selective ligand function in vivo , and that features outside the known binding interface contribute to their differences .
A small number of signaling systems control how an animal develops from a single cell into a complex organism made up of many different cell types . Signals pass back and forth between cells , switching genes on and off to direct the development of tissues and organs . One of these signaling systems , called Notch , is so ancient that it appears in nearly all multicellular organisms . A cell sends a Notch signal using proteins called Delta or Jagged ligands that span membrane of the cell , so that part of the protein sits inside the cell and part remains outside . To change the behavior of another cell , the ligands bind to proteins called Notch receptors that span the membrane of the receiving cell . Mammals have two types of Delta ligand , two types of Jagged ligand and four types of Notch receptor . Cells in different tissues display different combinations of these eight proteins . Two Delta ligands called DLL1 and DLL4 often appear together in developing organisms . Some tissues need both and some only the one or the other . In some cases one ligand can compensate if the other is missing , but in others not . It was not clear why this is , or which parts of the proteins are responsible . Tveriakhina et al . used mouse cells to investigate how DLL1 and DLL4 interact with two Notch receptors , called NOTCH1 and NOTCH2 . The results of these experiments show that while DLL1 can bind and activate both Notch receptors equally , DLL4 prefers to partner with NOTCH1 . To find out which parts of the ligands are responsible for this selectivity , Tveriakhina et al . created hybrid ligands that contained a mixture of regions from DLL1 and DLL4 . These suggest that the different binding preferences depend on parts of the ligands that sit outside cells and that lie outside the known sites of binding contact with the Notch receptors . Further experiments studied mice that had been engineered to produce hybrid ligands as replacements for DLL1 . A hybrid ligand consisting of the part of DLL1 that sits outside cells and the part of DLL4 found inside cells generated Notch signals in the tissue that depended on the activity of DLL1 . However , a hybrid consisting of the part of DLL4 that sits outside cells and the part of DLL1 found inside cells did not , showing that in developing mice the parts that sit outside the cells contribute to the different functions of DLL1 and DLL4 . Overall , the results presented by Tveriakhina et al . show that interactions between specific ligands and receptors play important roles in how mammals develop . Further efforts to understand which parts of the ligands affect selectivity could ultimately allow researchers to develop ways to modify how ligands and receptors interact . Such “molecular engineering” strategies could enable cell responses to be precisely controlled by pairing designer ligand-receptor pairs to develop cell-based therapies .