Neuralized-like proteins differentially activate Notch ligands
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Abstract
Notch signalling is a major signalling pathway coordinating cellular processes between neighbouring animal cells. In Drosophila, two ubiquitin ligases, Neuralized (Neur) and Mindbomb1 (Mib1), regulate Notch ligand activation and are essential for development. However, the mammalian orthologs of Neur, Neuralized-like (NEURL) 1 and 2, do not appear to be crucial for development, as double knock-out mice show no developmental defects. Thus, it is unclear if and how NEURL proteins regulate the four mammalian Notch ligands. To address these questions, we examined NEURL proteins’ ability to activate Notch ligands in humanized Drosophila and mammalian cell culture. We found that, unlike MIB1, NEURL proteins activate Notch only with a subset of mammalian ligands, which contain a Neuralized binding motif. This motif has the consensus sequence NxxN, present only in Notch ligands DLL1 and JAG1, but not in DLL4 and JAG2. Overall, we show that NEURL proteins activate specific Notch-ligands, suggesting a differential regulatory mechanism of Notch activation in mammals, which can potentially explain the limited role of NEURL proteins in mammalian development and homeostasis.
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Reviewer #1 (Evidence, reproducibility and clarity (Required)):
This is an interesting manuscript from two groups of experts in Notch signaling biology with complementary expertise in Drosophila genetics (Klein) and in biophysical studies of the Notch pathway (Sprinzak). The paper provides a cutting-edge structure-function dissection of the E3 ubiquitin ligase Neuralized and its mammalian homologs, Neurl1a and Neurl1a. The work is particularly relevant since the functions of mammalian Neurl1a and Neurl1b have been questioned, and more subtle altogether than those of fly Neuralized (as summarized by the authors in Fig. 1C). This is in part due to the …
Note: This response was posted by the corresponding author to Review Commons. The content has not been altered except for formatting.
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Reply to the reviewers
Reviewer #1 (Evidence, reproducibility and clarity (Required)):
This is an interesting manuscript from two groups of experts in Notch signaling biology with complementary expertise in Drosophila genetics (Klein) and in biophysical studies of the Notch pathway (Sprinzak). The paper provides a cutting-edge structure-function dissection of the E3 ubiquitin ligase Neuralized and its mammalian homologs, Neurl1a and Neurl1a. The work is particularly relevant since the functions of mammalian Neurl1a and Neurl1b have been questioned, and more subtle altogether than those of fly Neuralized (as summarized by the authors in Fig. 1C). This is in part due to the dominant effects of the E3 ubiquitin ligase Mindbomb1 (Mib1) in Notch ligand-expressing cells from mammalian systems. The authors use careful structure-function work in fly development (mostly wing imaginal discs) and in mammalian cell culture systems, including a clever approach to study the function of mammalian Neurl1a and Neurl1b and mammalian/fly Notch ligand hybrids in Drosophila to draw new conclusions about the function of Neurl1a/b, showing that they can function as activators of Notch signaling mediated by the Notch ligands Dll1 and Jag1, and not by Dll4 and Jag2, tracing these differential effects to the recognition of a short NXXN consensus sequence in the N-terminal region of the ligand's intracellular domain.
__response: __We thank the reviewer for highlighting the novelty of our findings and experimental approach.
Specific questions: -The current title of the manuscript is not very information-rich and would not allow a reader to gather key information about the findings without reading at least the abstract. Could this be improved? For example, by referring to differential activation of individual Notch ligands, or some other more direct description of the key findings?
__Response: __We appreciate the reviewer's suggestion; however, we believe that the general nature of the title is appropriate in this case.
-The authors design most key experiments documenting agonistic effects of Neurl1a/1b in a Mib1-deficient background, both in flies and in cell culture systems. This is understandable experimentally to isolate Neurl1a/b's effects in these experimental systems. However, this leaves open questions as to the prevailing effects of Neurl1a/b in cells that also express Mib1 (which the authors comment on in the discussion based on past findings, including some suggesting that Neurl1a/1b can function as Notch inhibitors through a ligand ubiquitination mechanism that may differ from their activating function).
Do the authors actually have data that could shed light on this discussion? For example, have they performed cell coculture assays in which Neurl1a or Neurl1b is co-expressed with a Notch ligand, but in the presence of Mib1? This condition seems to be systematically omitted from all the coculture experiments that are presented. It would be interesting to evaluate the net effect of Neurl1a/Neurl1b expression in a Mib1-sufficient system as well.
Response: We have systematically removed MIB1 in our experiments because it activates all ligands, making its removal necessary to show the differential activity of Neurls. The question regarding competition between Mib1 and Neurls, as highlighted by the reviewer, is indeed intriguing. However, systematically investigating this competition would require varying the relative levels of the two proteins in a controlled manner, which is beyond the scope of this manuscript.
That said, we will perform the competition experiments suggested by the reviewers (co-expressing ligands with both Neurl1 and Mib1) and test their activity as controls. While these experiments may provide some insight into the competition, they will not comprehensively address the entire topic.
-The paper suggests important predictions about mammalian functions of Neurl1a/1b, including the neurological effects that have been reported, in double-deficient mice, namely that that there are cells that only express Neurl1a/1b and not Mib1 and do rely on Dll1 and Jag1 for signaling. Could the authors at least comment on this prediction? Are there are any single cell atlases where candidate cells like that can be identified? Or would the authors predict that Neurl1a/1b could actually function as Notch agonist even in cells expressing Mib1? (see also previous comment)
Response: This is an interesting suggestion. We will try to find if we can identify any specific expression patterns of E3 ubiquitin ligases across different tissues.
-Some minor typos: line 305 should likely read "flies homozygous for (...)". Line 408, "for providing" repeated twice.
Response: We thank the reviewer for pointing out this typo.
Reviewer #1 (Significance (Required)):
Thank you for the opportunity to review this lovely collaborative paper. As indicated in my comments to the authors, the findings provide novel structure-function information about an understudied aspect of Notch signaling and clarify conflicting past data about the mammalian homologs of fly Neuralized. The approach is elegant and multidisciplinary, notably in regards to the combination of cell co-culture systems and Drosophila as a platform to study mammalian Neuralized proteins and hybrid Notch ligand molecules. The findings will be interesting to the field and will generate discussion. I would suggest that some additional information would be a plus to substantiate predictions about mammalian functions of Neurl1a/b, and also to clarify its effects in the presence or absence of concomitant Mib1 expression.
We thank the reviewer for their positive evaluation of our work and for suggesting potential future direction regarding the concomitant expression of Mib1 and Neurls proteins.
Reviewer #2 (Evidence, reproducibility and clarity (Required)):
Summary
The manuscript describes an analysis of specificity of functional interactions between mammalian Neuralized proteins and different human ligands for Notch. To investigate this, the authors take the approach of constructing hybrid proteins that contain the intracellular domain of the human ligands and the extracellular domain of the Drosophila Delta or Serrate, and investigate their activity in vivo, in the Drosophila wing disc. The latter is a well-established model tissue for assessing Notch ligand activity. As a second assay they express mammalian neutralized constructs in human cells for luciferase-based Notch signal reporter assays. The experiments are well presented and described and make a strong case for the conclusions that both Neurl1 and 2 can activate Notch signalling by Dll1 and Jag1 but not Dll4 and Jag2. Use of different mutant intracellular domains is used to show the importance of the NXXN motif, which in Drosophila is required for Neuralized interaction with Delta and Serrate. The use of missense mutations and in particular the reactivation of the cryptic NXXD site in Dll4 by substitution to N is convincing for establishing the importance of the motif. There is also colocalization data to support the conclusion that there is likely to be NXXN-dependent complex formation between the ligand and Neuralized proteins. This latter conclusion would be made firmer fi there were pull down data to support it, although to be fair it is most unlikely that another explanation, other than complex formation could account for the observation of both colocalization and ligand activation.
__Response: __We appreciate the reviewer's positive assessment of our manuscript and their support for the conclusions drawn from our experiments. We intend to conduct the suggested co-IP experiments with our cell culture assays to further supplement our current data.
__ Major comments__ The main limitation of the work is that it is mostly based on overexpression of constructs to activate ectopic expression rather than gene editing endogenous genes. It would be helpful if the authors could comment on the limitations of the work in discussion.
Two points of data included in the work are important in mitigating this limitation. Firstly, the experiments in the wing disc and cell culture are taking place in a mindbomb mutant background and the activation is observed is therefore a rescue of activity that has been lost.
Secondly, and importantly, the final experiment makes use of a Dl mutant Drosophila line which shows embryo lethality when homozygous, with the characteristic neurogenic phenotype. Rescue of lethality can be brought about by knock-in experiments which restore Dl function and this is also true for the ligand hybrid constructs that introduce mammalian ligand intracellular domains only when they include the NXXN motif This indicates the importance of the motif in normal development- Overall, the data presented in the paper is convincing as regards the conclusions made.
__Response: __We thank the reviewer for their very positive evaluation and his constructive suggestions, which have helped to improve the manuscript. In line with these suggestions, we will include additional data analyzing the bristle SOP selection, a process dependent on Neur. Our Results show that homozygosity of the DlattP-Dl-DLL1 allele, but not the DlattP-Dl-DLL4 allele, leads to correct Notch mediated selection. This finding provides further evidence that Neur requires the NxxN motif in the ICD of a ligand to activate DSL ligands. Notably, we previously showed that this selection relies on the NxxN motif of Dl (Troost et al., 2023). We will further emphasize in the discussion the ability of Dl-DLL4 hybrid ligands, containing a reconstructed NxxN motif, to rescue the neurogenic phenotype of Dl mutants.
Minor points In figure 1 the legend for D says that cryptic sites are substitutions of N for E or Q, but the figure and main text indicate that the substitutions are N to E or D.
Response: We thank the reviewer for pointing this out. We will correct this mistake.
In the remain figures it would be helpful to include in the figure legends and indications of the numbers of wing discs, embryos for which the images shown are representative of.
__Response: __We will quantify the experiments conducted in the wing imaginal discs of Drosophila by measuring the wing field size along the dorsal-ventral axis relative to the anterior-posterior axis. Statistical analysis will be performed to demonstrate statistical significance across n=5 experiments for each sample.
In Fg 3 The activation of Notch, by neural1 and Dl-Jag1 in B'" is stronger in the ventral side of the disc than the dorsal whereas, although activation of the same ligand by Neurl2 in C'" is weaker the majority of the ectopic wingless expression is on the dorsal compartment. Is there any reason for the switch in preference between the two neutralized proteins? Overgrowth of the wing disc seems to be similar on both sides and so am wondering if the picture is representative of the ectopic wingless distribution in this case.
Response: As discussed above we will perform quantification and statistical analysis across multiple experiments to confirm that our images are truly representative.
Reviewer #2 (Significance (Required)):
Significance
Previous work on double genetic knockouts of the two mouse Neuralized genes cast doubt as to whether Neuralized proteins play a role in Notch signal activation in mammals, unlike in Drosophila. There is, however, some genetic indications that spatial memory requires both Notch and neutralized proteins and may represent a specialised function limited to the Neuralized interaction. There are likely to be more subtle contexts waiting to be uncovered. The work is therefore showing important proof of principle for establishing the functionality of the mammalian Neurl proteins and highlights new findings indicting specialisation of the different ligands for interactions with Notch components. Elucidation of such specialisations will help understand why the diversity of different homologues of Notch and ligand have evolved and are maintained in the vertebrate genome compared to the single Notch and two ligands in Drosophila. Since Notch and it misregulation are widely involved in development, health and disease and there is much interest in developing therapeutic interactions that alter Notch activity then the work is likely of broad interest.
We thank the reviewer for the very positive evaluation and his useful suggestions which were helpful in improving the manuscript.
Reviewer #3 (Evidence, reproducibility and clarity (Required)):
**Summary**
Notch signalling is one of the major evolutionarily conserved signalling pathways involved in numerous developmental, physiological and pathological processes. Activation of the Notch receptor first requires ubiquitination of its ligands (collectively temed DSL), leading to a 'pulling force" that, upon ligand-receptor engagement, exposes Notch to intramembrane proteolysis leading to the nuclear translocation of the receptor's intracellular domain and activation of target genes with its DNA-binding co-factors.
While both Neuralized (Neurl) and Mind bomb are the E3 ubiquitin ligases for Notch ligands required for Drosphila development, in mammals, the Neur homologues Neur1 (officially Neurl1) and Neur2 (officially Neurl1B) are dispensable for development since double Neur1/2 knock-out mice have no developmental phenotype (but both Neur homologues are involved the the memory-related functions of Notch pathway in adulthood). Rather, just one of the two mammalian Mind bomb homologues, Mib1, functions as the chief E3 ligase for Notch ligands during mammalian development as evidenced by its Notch-related knockout phenotype.
Therefore, it has not been fully established whether and how the NEUR proteins regulate the mammalian Notch ligands. To clarify this issue, the authors assessed the capability of Drosophila Neur and mammalian NEUR1 and 2 proteins to activate the various hybrid Notch ligands (containing extracellularly Drosophila Delta and intracellularly the various Notch ligands' intracellular domains) in Drosophila wing dics and mammalian cell culture. The authors found that NEUR proteins only activate the Notch ligands containing a Neuralized binding motif, with the consensus sequence NxxN, that is present in DLL1 and JAG1, but not in DLL4 and JAG2. The authors also analyse the intracellular domains of mammalian Notch ligands DLL1, DLL4, JAG1 and JAG2 in Drosophila by generating knock-in alleles where endogenous Dl expression had been substituted for those of hybrid Notch ligands. This analysis showed that only in Dl-DLL1 and Dl-JAG1 flies but not in Dl-DLL4 and Dl-JAG" flies is the embryonic lethality rescued, the results being in agreement with the hybrid Dl-DLL experiments on wing dics reported earlier in this work.
The authors conclude that their findings suggest that the activation mechanism of Notch during development differs between Drosophila (where both Neur and Mib1 are required for Notch-related developmental processes ) and mammals and that this could possibly explain the apparently lesser relevance of mammalian NEUR proteins for developmental Notch signalling.
*Evidence and clarity*
The manuscript is quite laconic but clearly written. The evidence presented by the authors, given the heterologous and in vitro nature (i.e using mammalian or hybrid Notch ligands and mammalian E3 ligases thereof in Drosophila and cell cultures) of the study is generally trustworthy but limited in the sense that it probably does not allow definitive conclusions to be drawn as to the differing nature of the action of the E3 ligases of Notch ligands in flies vs mammals in vivo.
__Response: __We thank the reviewer for their positive evaluation of our work and their constructive criticism. We would like to clarify that we do not conclude that the activation mechanism differs between mammals and flies. Our findings demonstrate that the signalling mechanisms of fly Neur and mammalian Neurl's follow the same fundamental rules. Moreover, our study does not aim to provide a definitive answer to how signalling differs between species. Instead, we utilized the 'humanized fly' system to show that Neurl proteins specifically activate Dll1 and Jag1, but not Dll4 and Jag2, which lack a neuralized binding site.
*Reproducibility*
As will be mentioned a number of times, these reviewers would like to enquire as to the reasons for not providing a statistical analysis of variation in the fly wing disc-based experiments (where the readout was either resuce of Wg expression or induction of ectopic Wg expression).
Response: We thank the reviewer for raising this important point. As outlined below, we will quantify the fly experiments and conduct statistical analysis across multiple experimental datasets to further substantiate our claims.
Also, while the constructs used in the study were inserted into the same genomic landing sites to achieve comparable levels of expression of the various proteins, these reviewers would like to see data on the levels of expression of NEUR1 and 2 as well as the hybrid Notch ligands.
**Major comments**
Comment on fly wing disc experiments:
The authors study both the capability of two different mammalian E3 ubiquitin ligases, Neuralized-like 1 and 2 (mouse Neur1 and human NEUR2) to activate four different Notch receptors (DLL1 and 2, JAG1 and 2) in flies and mammalian cell culture system. In flies, they first analyse the capability of Drosophila Neur (as a positive control) and Neur1 and NEUR2 to activate the various Notch ligands (based on wingless activation as a readout) in wild-type wings (where, Mind bomb 1, or Mib1 is the only E3 ligase for Notch ligands present) and Mib1 mutant wing discs (which lack any E3 ligands of Notch receptors). The authors then test four humanised, hybrid Notch ligands (all five N ligands bar Dll3 since the latter does not transactivate the Notch receptor) - where mammalian Notch ligands' intracellular domains, or ICDs, have been attached to fly Dl (Dl-Dll1, Dl-Dll4, Dl-JAG1, Dl-JAG2) - for their capacity to mediate Mib1-dependent activation of Notch (with ectopic Wg expression in wing discs as its readout). They found that all 4 ligands can activate Nocth in wild-type wings (where Mib1 is present), with Dl-JAG2 being less effective than the other 3 hybrid ligands, implying that such hybrid, humanised ligands can be usd in studying Notch pathway activation in Drosophila (thereby constituting a mixed/heterologous experimental system). The reviewers would like to get a comment as to the reason for the weaker activity of Dl-JAG2 in this set-up?.
Response: We do not have a definitive answer as to why the ICDs differ in their activity within MIb1-dependent signalling, since this question was not addressed in the scope of this work. However, it our findings demonstrate that the hybrid ligands are functional in Drosophila and that their differential behavior in Neur-mediated signaling is not attributed to a trivial explanation, e. g. that the hybrid ligands generally display no activity. There are several potential explanations for these differences. One possibility is variations in position, arrangement, or number of targeted lysines among the ICDs. These lysines serve as substrates for ubiquitylation and determine the rate of endocytosis, which in turn impacts the signaling activity of the corresponding ligand/hybrid. Another plausible explanation is differences in affinity of the binding sites of Mib1, which would similarly result in variations in ubiquitylation and endocytosis rates. Regardless, we emphasize that resolving this question does not affect any of the conclusions of the manuscript.
Also, the reviewers would like to get a comment as to why was not a Neur mutant set-up used, only Mib1 mutant dics?
Response: Neur is only expressed at a very late stage in wing development and is restricted to specific single cells (sensory organ precursors). Consequently, even if mutants were present, their impact would be limited to these cells. Moreover, the Neur promoter has a highly complex architecture, which makes it exceedingly difficult to manipulate for experiments involving this mutation. We will address these considerations in the revised manuscript.
The authors then found that only two of these hybrid ligands - Dl-DLL1 and Dl-JAG1 but not Dl-DLL4 or Dl-JAG2 - can be used to activate Notch in the above wing assay when Mib1 was mutant. This is consistent with the fact that the NxxN-based Neuralized Binding motif (NBM) is present in DLL1 and JAG1 only. Using the wing paradigm, the authors also show by either mutating the full NBM (NxxN) in DLL1 or changing the cryptic, "weak" NBM in DLL4 (containing NxxD sequence) into "full/strong" NxxN one that the NBM in the various Notch ligands is required and sufficient for activation of the Notch pathway.
Overall, the fly experiments are convincing in showing diffrential activation of Notch ligands. However, no statistical analysis of the experimental variation in these studies - neither for the number of wing discs analysed per (hybrid) Notch ligand tested nor the extent of a given experimental manipulation's effect is included. We deem that if the images presented in Figures 2 and 3 are truly representative, this needs to be made explicitly clear.
Response: We thank the reviewer for their positive evaluation of our work and for the constructive comments, which we will consider and include into the manuscript. While we have repeated all experiments with multiple flies, we acknowledge the critique regarding the absence of statistical analysis.
To address this, we will quantify the experiments conducted in the wing imaginal discs of Drosophila. We will do that by measuring the wing field size along the dorsal-ventral axis relative to the anterior-posterior axis. We will perform statistical analysis to assess the statistical significance between experiments, using data from n=5 experiments for each sample.
Comment on fly embryonic Delta neurogenic phenotype's rescue experiments by replacing Dl with the hybrid ligands: The authors analysed the capacity of the ICDs of the mammalian ligands to rescue the Dl phenotype in Drosophila, ie. their activation capability at the organismal level. This was achieved by generating knock-in alleles expressing the hybrid ligands in place of Dl. The notion that only NBM-containing hybrid ligands was strengthened by this analysis since it showed that only NBM-containing hybrid ligands - Dl-DLL1 and Dl-JAG1 - but not Dl-DLL4 nor Dl-JAG2 rescued the Dl neurogenic embryonic lethal phenotype. Since this experimental set-up relied on the endogoneous Drosophila E3 ligases for activating the Notch ligands, the capacity of mammalian NEUR1 and 2 proteins to complement the capacity of the hybrid ligands to activate Notch to activate these ligands was not addressed. Please comment as to the reasons for this apparent omission and if such an analyis lies beyond the scope of current work, what would be the expected results of such experiment in the light of other experiments conducted in the course of this work?
Response: Testing whether mammalian Neurl1 and Neurl2 can replace Drosophila Neur in an endogenous setting is an intriguing question; however, it falls outside the focus of this study. Performing such an experiment would be highly challenging due to complex and not well understood architecture of Neur gene in Drosophila. Additionally, we believe it is highly unlikely that the mammalian NEURLs proteins would fully compensate for the loss of function in a Drosophila Neur mutant.
Journal-agnostic peer review: evaluate the paper as it stands independently from potential journal fit.
Are the claims and the conclusions supported by the data or do they require additional experiments or analyses to support them?
Generaly yes, put please see the above comments on the absence of statistical analysis of reproducibility/ variation (if any) in fly wing disc experiments.
**Reviewer's additional recommendations:**
To publish in a higher-ranking journal, the co-localisation analyses of Notch ligands and its various E3 ubiquitin ligases studied probably needs to be replaced by a more rigorous, ideally FRET-based approach.
Response: We thank the reviewer for the comment. The co-localization assay is quite a robust and functional approach, as it provides clear evidence that endocytosis into a different compartment has occurred with functional ligands, as opposed to non-functional ligands. This serves as a quantitative and rigorous indicator for functional differences between these ligand types.
Nevertheless, we acknowledge that co-localization is not a direct measure of molecular interactions between Neurl1 and Notch ligands. To address this, as suggested by the reviewer, we will perform co-IP to show the differential interaction between Neurl1 and specific Notch ligands. Additionally, we will attempt a proximity ligation assay (PLA), which we consider to be a more direct and suitable method for detecting interactions between NEURLs and Notch ligands in this context, compared to FRET.
Since previous studies have shown that the Notch ligands are (mostly) poly- or mono-ubiquitylated by the E3 ubiquitin ligases Mib and the NEUR proteins, ideally, this or its follow-up study would benefit from analysis of the ubiquitylation status of the various hybrid Notch ligands.
Response: We thank the reviewer for the suggestion. The ubiquitylation pattern by Neurl1 is beyond the topic of the current manuscript.
Also, it would be useful to show the strength of interaction between the hybrid Notch ligands and NEUR1 and NEUR2 by ising a co-immunoprecipitation based approach.
Response: As suggested by the reviewer, we plan to perform co-IP and/or PLA to show the differential binding of NEURL1 to the different ligands. However, due to the observed toxicity of NEURL2 in our cells, it has been excluded from our assays.
Please request additional experiments only if they are essential for the conclusions. Alternatively, ask the authors to qualify their claims as preliminary or speculative, or to remove them altogether.
These reviewers do not strictly request any further rexperiments. However, since the mammalian NEUR2 could not be studied in cell cultures of U2OS cells due to its toxicity, we would like the auhtors to explain the choice of this cell line. Perhaps a cell line whose viability is not impaired by NEUR2 should be (or should have been) used?
Response: The decision not to use other cell lines was based on several strict experimental requirements. The most stringent requirement was the need to generate a MIB1 knockout cell line, as MIB1 strongly activates all ligands. The availability of having MIB1 KO U2OS cells enabled these experiments.
If you have constructive further reaching suggestions that could significantly improve the study but would open new lines of investigations, please label them as "OPTIONAL".
As mentioned above, the NEUR2's capacity to activate the hybrid ligands in U2OS cells could not be addressed to due to its toxicity. A more optimal cell line will have to be used in follow-up studies.
Also, these findings ultimately warrant in vivo studies using mice to definitively ascertain whether they also hold equally true there.
Are the suggested experiments realistic in terms of time and resources? It would help if you could add an estimated time investment for substantial experiments.
The suggested experiments are optional apart from statistical analysis of variation (if any) in the fly wing disc experiments. If there is no (apparently significant) variation in these data, this needs to explicitly stated.
Response: We thank the reviewer for their thoughtful assessment. We will conduct the requested statistical analysis and perform some of the suggested supporting experiments as detailed in the response.
Are the data and the methods presented in such a way that they can be reproduced?
Generally yes, but see above about the lack of statistical data on the variation (if any).
Are the experiments adequately replicated and statistical analysis adequate?
Generally yes, but again, please see above about the lack of statistical data on the variation (if any).
**Minor comments**
Comment#1 (on the abstract and introduction):
In the Abstract, the authors state that there are four Notch ligands in mammals (lines 21 and 22): "Thus, it is unclear how NEURL proteins regulate the four mammalian Notch ligands". In the Introduction, they correctly state that there are five Notch ligands in mammals (lines 38 and 39): „In mammals, there are five ligands, three from the Delta-like (Dll) family (Dll1, Dll3, Dll4), and two from the Jagged (Jag) family (Jag1 and Jag2)." There are five Notch ligands in mammals (Dll1, Dll3, Dll4, Jag1, Jag2), and it is obvious that the authors are very well aware of this (they state in lines 146-147): "We excluded the ICD of DLL3 since it is not a ligand capable of trans-activation of Notch" (the four ligands included were Dll1, Dll4, Jag1 and Jag2)." Therefore, a claricifaction is required in the part of Abstract (i.e lines 21 ansd 22) - did the authors mean the four mammalian Notch ligands they actually studied (i.e Dll1, Dll4, Jag1, Jag2) or is there an oversight and the auhtors actually intended to write "the five Nocth ligands in mammals".? In either case, a correction is required in this reviewer's opinion.
Response: We are fully aware of this point, and will address it by providing clarification in the abstract as suggested.
Specific experimental issues that are easily addressable.
NEUR2 could not be studied in mammalian cell cultures due to its toxicity in the U2OS cell line, the one used by the authors. The use of another cell line would not be probably overly time-consuming; however, if this experiment lies outside the scope of current work, we would like to hear the authors' comment on this matter.
Response: This is addressed above.
Are prior studies referenced appropriately? Generally yes, but four prior studies go unmentioned: the two 2001 mouse Neur1 knock-out studies reporting no Notch-like developmental phenotype (Ruan et al, PNAS; Vollrath et al, Mol Cell Biol), the 2002 study of mouse, rat and human NEUR1 expression, subcellular localisation (Timmusk et al, Mol Cell Neuroscience) and the 2009 cell culture-based study of NEUR2's interaction with DLL1 and DLL4 (Rullinkov et al, BBRC). The non-requirement of NEUR1 and 2 proteins in mammalian developmental Notch signalling could partly be explained by the fact that NEUR1 is not highly expressed during mouse embryonic/foetal development - its expression becomes considerably more pronounced only postnatally (Timmusk et al, 2002).
Response: We will incorporate these references into the introduction and discuss the low expression of Neurls during development as a possible reason for the non-requirement in this context.
Are the text and figures clear and accurate?
Yes. These reviewers find the cartoon-based explanations of the experimental set-up in each figure helpful for enhancing the manuscript's overall clarity.
Response: We thank the reviewers for the positive feedback!
Do you have suggestions that would help the authors improve the presentation of their data and conclusions?
Please see above about the lack of statistical data on the variation (if any) in fly wing dic experiments and referencing of the 4 papers that are currently excluded.
Response: These will be corrected in the revised version.
Reviewer #3 (Significance (Required)):
- Significance Provide contextual information to readers (editors and researchers) about the novelty of the study, its value for the field and the communities that might be interested. The following aspects are important: General assessment: provide a summary of the strengths and limitations of the study. What are the strongest and most important aspects? What aspects of the study should be improved or could be developed? This study uses the amenability of Drosophila to study the mammalian NEUR proteins' (NEUR1 and NEUR2) activity upon Notch ligands using hybrid Notch ligands containing mammalian ICDs (intracellular domains) fused to the extracellular domain of Drosophila Delta (Dl). It confirms and extends prior studies showing that Notch ligands can be (strongly) activated only by the E3 ubiquitin ligases containing the Neuralized Binding Motif (NBM).
Response: We respectfully disagree with the reviewer's assessment on this point. Our study is the first to demonstrate that Neurl proteins differentially activate Dll1 and Jag1, but not Dll4 and Jag2. This findings is further supported by the significance comments of the other reviewers.
However, since this study was based on using hybrid ligands containing mammalian ICDs of Notch ligands fused to the extracellular domain of Drosophila Delta (Dl), it is somewhat artificial. While NEUR1 was also studied in mammalian cell cultures (but not NEUR2 due to its toxicity), only an in vivo study using mice expressing with systematic changes to the Notch ligands' NBM will definitively reveal whether the conclusions reached by the authors hold true in vivo in a non-heterologous system.
Response: We firmly believe that our combined 'humanized fly' model and quantitative cell culture assay represents an innovative and rigorous approach for testing humanized proteins in in-vivo settings, without the need for extensive mouse genetics. The conclusions of our experiments should not be dismissed solely on the grounds of "not being performed in mice," as this would undermine much of current scientific research.
Advance: compare the study to the closest related results in the literature or highlight results reported for the first time to your knowledge; does the study extend the knowledge in the field and in which way? Describe the nature of the advance and the resulting insights (for example: conceptual, technical, clinical, mechanistic, functional,...). The study's advances are chiefly mechanistic and functional since they show more definitively that the reason underlying the differing activation of four mammalian Notch ligands by mammalian NEUR1 and NEUR2 is mostly based upon the presence or otherwise of a conserved Neuralized Binding Motif, NBM. Audience: describe the type of audience ("specialised", "broad", "basic research", "translational/clinical", etc...) that will be interested or influenced by this research; how will this research be used by others; will it be of interest beyond the specific field?
The audience for this study is the research studying the Notch signalling pathway. Since dysregulation of this pathway is implicated in a number of devastating diseases, any improved understanding of its mechanistic underpinnings could in the long run lead to better therapeutic management of diseases with significant involvement of malfunctioning Notch signalling.
Please define your field of expertise with a few keywords to help the authors contextualize your point of view. Indicate if there are any parts of the paper that you do not have sufficient expertise to evaluate. Molecular biology, molecular neuroscience, developmental biology, cell-cell signalling, Notch signalling. All parts of the manuscript fall within our expertise.
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Referee #3
Evidence, reproducibility and clarity
Summary
Notch signalling is one of the major evolutionarily conserved signalling pathways involved in numerous developmental, physiological and pathological processes. Activation of the Notch receptor first requires ubiquitination of its ligands (collectively temed DSL), leading to a 'pulling force" that, upon ligand-receptor engagement, exposes Notch to intramembrane proteolysis leading to the nuclear translocation of the receptor's intracellular domain and activation of target genes with its DNA-binding co-factors.
While both Neuralized (Neurl) and Mind bomb are the E3 ubiquitin ligases for Notch ligands required for Drosphila …
Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.
Learn more at Review Commons
Referee #3
Evidence, reproducibility and clarity
Summary
Notch signalling is one of the major evolutionarily conserved signalling pathways involved in numerous developmental, physiological and pathological processes. Activation of the Notch receptor first requires ubiquitination of its ligands (collectively temed DSL), leading to a 'pulling force" that, upon ligand-receptor engagement, exposes Notch to intramembrane proteolysis leading to the nuclear translocation of the receptor's intracellular domain and activation of target genes with its DNA-binding co-factors.
While both Neuralized (Neurl) and Mind bomb are the E3 ubiquitin ligases for Notch ligands required for Drosphila development, in mammals, the Neur homologues Neur1 (officially Neurl1) and Neur2 (officially Neurl1B) are dispensable for development since double Neur1/2 knock-out mice have no developmental phenotype (but both Neur homologues are involved the the memory-related functions of Notch pathway in adulthood). Rather, just one of the two mammalian Mind bomb homologues, Mib1, functions as the chief E3 ligase for Notch ligands during mammalian development as evidenced by its Notch-related knockout phenotype.
Therefore, it has not been fully established whether and how the NEUR proteins regulate the mammalian Notch ligands. To clarify this issue, the authors assessed the capability of Drosophila Neur and mammalian NEUR1 and 2 proteins to activate the various hybrid Notch ligands (containing extracellularly Drosophila Delta and intracellularly the various Notch ligands' intracellular domains) in Drosophila wing dics and mammalian cell culture. The authors found that NEUR proteins only activate the Notch ligands containing a Neuralized binding motif, with the consensus sequence NxxN, that is present in DLL1 and JAG1, but not in DLL4 and JAG2. The authors also analyse the intracellular domains of mammalian Notch ligands DLL1, DLL4, JAG1 and JAG2 in Drosophila by generating knock-in alleles where endogenous Dl expression had been substituted for those of hybrid Notch ligands. This analysis showed that only in Dl-DLL1 and Dl-JAG1 flies but not in Dl-DLL4 and Dl-JAG" flies is the embryonic lethality rescued, the results being in agreement with the hybrid Dl-DLL experiments on wing dics reported earlier in this work. The authors conclude that their findings suggest that the activation mechanism of Notch during development differs between Drosophila (where both Neur and Mib1 are required for Notch-related developmental processes ) and mammals and that this could possibly explain the apparently lesser relevance of mammalian NEUR proteins for developmental Notch signalling.
Evidence and clarity
The manuscript is quite laconic but clearly written. The evidence presented by the authors, given the heterologous and in vitro nature (i.e using mammalian or hybrid Notch ligands and mammalian E3 ligases thereof in Drosophila and cell cultures) of the study is generally trustworthy but limited in the sense that it probably does not allow definitive conclusions to be drawn as to the differing nature of the action of the E3 ligases of Notch ligands in flies vs mammals in vivo.
Reproducibility
As will be mentioned a number of times, these reviewers would like to enquire as to the reasons for not providing a statistical analysis of variation in the fly wing disc-based experiments (where the readout was either resuce of Wg expression or induction of ectopic Wg expression). Also, while the constructs used in the study were inserted into the same genomic landing sites to achieve comparable leves of expression of the various proteins, these reviewers would like to see data on the levels of expression of NEUR1 and 2 as well as the hybrid Notch ligands.
Major comments
Comment on fly wing disc experiments:
The authors study both the capability of two different mammalian E3 ubiquitin ligases, Neuralized-like 1 and 2 (mouse Neur1 and human NEUR2) to activate four different Notch receptors (DLL1 and 2, JAG1 and 2) in flies and mammalian cell culture system. In flies, they first analyse the capability of Drosophila Neur (as a positive control) and Neur1 and NEUR2 to activate the various Notch ligands (based on wingless activation as a readout) in wild-type wings (where, Mind bomb 1, or Mib1 is the only E3 ligase for Notch ligands present) and Mib1 mutant wing discs (which lack any E3 ligands of Notch receptors). The authors then test four humanised, hybrid Notch ligands (all five N ligands bar Dll3 since the latter does not transactivate the Notch receptor) - where mammalian Notch ligands' intracellular domains, or ICDs, have been attached to fly Dl (Dl-Dll1, Dl-Dll4, Dl-JAG1, Dl-JAG2) - for their capacity to mediate Mib1-dependent activation of Notch (with ectopic Wg expression in wing discs as its readout). They found that all 4 ligands can activate Nocth in wild-type wings (where Mib1 is present), with Dl-JAG2 being less effective than the other 3 hybrid ligands, implying that such hybrid, humanised ligands can be usd in studying Notch pathway activation in Drosophila (thereby constituting a mixed/heterologous experimental system). The reviewers would like to get a comment as to the reason for the weaker activity of Dl-JAG2 in this set-up?.
Also, the reviewers would like to get a comment as to why was not a Neur mutant set-up used, only Mib1 mutant dics? The authors then found that only two of these hybrid ligands - Dl-DLL1 and Dl-JAG1 but not Dl-DLL4 or Dl-JAG2 - can be used to activate Notch in the above wing assay when Mib1 was mutant. This is consistent with the fact that the NxxN-based Neuralized Binding motif (NBM) is present in DLL1 and JAG1 only. Using the wing paradigm, the authors also show by either mutating the full NBM (NxxN) in DLL1 or changing the cryptic, "weak" NBM in DLL4 (containing NxxD sequence) into "full/strong" NxxN one that the NBM in the various Notch ligands is required and sufficient for activation of the Notch pathway.
Overall, the fly experiments are convincing in showing diffrential activation of Notch ligands. However, no statistical analysis of the experimental variation in these studies - neither for the number of wing discs analysed per (hybrid) Notch ligand tested nor the extent of a given experimental manipulation's effect is included. We deem that if the images presented in Figures 2 and 3 are truly representative, this needs to be made explicitly clear. Comment on fly embryonic Delta neurogenic phenotype's rescue experiments by replacing Dl with the hybrid ligands: The authors analysed the capacity of the ICDs of the mammalian ligands to rescue the Dl phenotype in Drosophila, ie. their activation capability at the organismal level. This was achieved by generating knock-in alleles expressing the hybrid ligands in place of Dl. The notion that only NBM-containing hybrid ligands was strengthened by this analysis since it showed that only NBM-containing hybrid ligands - Dl-DLL1 and Dl-JAG1 - but not Dl-DLL4 nor Dl-JAG2 rescued the Dl neurogenic embryonic lethal phenotype. Since this experimental set-up relied on the endogoneous Drosophila E3 ligases for activating the Notch ligands, the capacity of mammalian NEUR1 and 2 proteins to complement the capacity of the hybrid ligands to activate Notch to activate these ligands was not addressed. Please comment as to the reasons for this apparent omission and if such an analsyis lies beyond the scope of current work, what would be the expected results of such experiment in the light of other experiments conducted in the course of this work? Journal-agnostic peer review: evaluate the paper as it stands independently from potential journal fit.
Are the claims and the conclusions supported by the data or do they require additional experiments or analyses to support them?
Generaly yes, put please see the above comments on the absence of statistical analysis of reproducibility/ variation (if any) in fly wing disc experiments.
Reviewer's additional recommendations:
To publish in a higher-ranking journal, the co-localisation analyses of Notch ligands and its various E3 ubiquitin ligases studied probably needs to be replaced by a more rigorous, ideally FRET-based approach. Since previous studies have shown that the Notch ligands are (mostly) poly- or mono-ubiquitylated by the E3 ubiquitin ligases Mib and the NEUR proteins, ideally, this or its follow-up study would benefit from analysis of the ubiquitylation status of the various hybrid Notch ligands. Also, it would be useful to show the strength of interaction between the hybrid Notch ligands and NEUR1 and NEUR2 by ising a co-immunoprecipitation based approach. Please request additional experiments only if they are essential for the conclusions. Alternatively, ask the authors to qualify their claims as preliminary or speculative, or to remove them altogether. These reviewers do not strictly request any further rexperiments. However, since the mammalian NEUR2 could not be studied in cell cultures of U2OS cells due to its toxicity, we would like the auhtors to explain the choice of this cell line. Perhaps a cell line whose viability is not impaired by NEUR2 should be (or should have been) used? If you have constructive further reaching suggestions that could significantly improve the study but would open new lines of investigations, please label them as "OPTIONAL". As mentioned above, the NEUR2's capacity to activate the hybrid ligands in U2OS cells could not be addressed to due to its toxicity. A more optimal cell line will have to be used in follow-up studies. Also, these findings ultimately warrant in vivo studies using mice to definitively ascertain whether they also hold equally true there.
Are the suggested experiments realistic in terms of time and resources? It would help if you could add an estimated time investment for substantial experiments.
The suggested experiments are optional apart from statistical analysis of variation (if any) in the fly wing disc experiments. If there is no (apparently significant) variation in these data, this needs to explicitly stated.
Are the data and the methods presented in such a way that they can be reproduced?
Generally yes, but see above about the lack of statistical data on the variation (if any).
Are the experiments adequately replicated and statistical analysis adequate?
Generally yes, but again, please see above about the lack of statistical data on the variation (if any).
Minor comments
Comment#1 (on the abstract and introduction):
In the Abstract, the authors state that there are four Notch ligands in mammals (lines 21 and 22):
"Thus, it is unclear how NEURL proteins regulate the four mammalian Notch ligands". In the Introduction, they correctly state that there are five Notch ligands in mammals (lines 38 and 39): „In mammals, there are five ligands, three from the Delta-like (Dll) family (Dll1, Dll3, Dll4), and two from the Jagged (Jag) family (Jag1 and Jag2)." There are five Notch ligands in mammals (Dll1, Dll3, Dll4, Jag1, Jag2), and it is obvious that the authors are very well aware of this (they state in lines 146-147): "We excluded the ICD of DLL3 since it is not a ligand capable of trans-activation of Notch" (the four ligands included were Dll1, Dll4, Jag1 and Jag2)." Therefore, a claricifaction is required in the part of Abstract (i.e lines 21 ansd 22) - did the authors mean the four mammalian Notch ligands they actually studied (i.e Dll1, Dll4, Jag1, Jag2) or is there an oversight and the auhtors actually intended to write "the five Nocth ligands in mammals".? In either case, a correction is required in this reviewer's opinion.Specific experimental issues that are easily addressable.
NEUR2 could not be studied in mammalian cell cultures due to its toxicity in the U2OS cell line, the one used by the authors. The use of another cell line would not be probably overly time-consuming; however, if this experiment lies outside the scope of current work, we would like to hear the authors' comment on this matter. Are prior studies referenced appropriately? Generally yes, but four prior studies go unmentioned: the two 2001 mouse Neur1 knock-out studies reporting no Notch-like developmental phenotype (Ruan et al, PNAS; Vollrath et al, Mol Cell Biol), the 2002 study of mouse, rat and human NEUR1 expression, subcellular localisation (Timmusk et al, Mol Cell Neuroscience) and the 2009 cell culture-based study of NEUR2's interaction with DLL1 and DLL4 (Rullinkov et al, BBRC). The non-requirement of NEUR1 and 2 proteins in mammalian developmental Notch signalling could partly be explained by the fact that NEUR1 is not highly expressed during mouse embryonic/foetal development - its expression becomes considerably more pronounced only postnatally (Timmusk et al, 2002).
Are the text and figures clear and accurate?
Yes. These reviewers find the cartoon-based explanations of the experimental set-up in each figure helpful for enhancing the manuscript's overall clarity.
Do you have suggestions that would help the authors improve the presentation of their data and conclusions?
Please see above about the lack of statistical data on the variation (if any) in fly wing dic experiments and referencing of the 4 papers that are currently excluded.
Significance
Provide contextual information to readers (editors and researchers) about the novelty of the study, its value for the field and the communities that might be interested. The following aspects are important:
General assessment: provide a summary of the strengths and limitations of the study. What are the strongest and most important aspects? What aspects of the study should be improved or could be developed? This study uses the amenability of Drosophila to study the mammalian NEUR proteins' (NEUR1 and NEUR2) activity upon Notch ligands using hybrid Notch ligands containing mammalian ICDs (intracellular domains) fused to the extracellular domain of Drosophila Delta (Dl). It confirms and extends prior studies showing that Notch ligands can be (strongly) activated only by the E3 ubiquitin ligases containing the Neuralized Binding Motif (NBM). However, since this study was based on using hybrid ligands containing mammalian ICDs of Notch ligands fused to the extracellular domain of Drosophila Delta (Dl), it is somewhat artificial. While NEUR1 was also studied in mammalian cell cultures (but not NEUR2 due to its toxicity), only an in vivo study using mice expressing with systematic changes to the Notch ligands' NBM will definitively reveal whether the conclusions reached by the authors hold true in vivo in a non-heterologous system.
Advance: compare the study to the closest related results in the literature or highlight results reported for the first time to your knowledge; does the study extend the knowledge in the field and in which way? Describe the nature of the advance and the resulting insights (for example: conceptual, technical, clinical, mechanistic, functional,...). The study's advances are chiefly mechanistic and functional since they show more definitively that the reason underlying the differing activation of four mammalian Notch ligands by mammalian NEUR1 and NEUR2 is mostly based upon the presence or otherwise of a conserved Neuralized Binding Motif, NBM.
Audience: describe the type of audience ("specialised", "broad", "basic research", "translational/clinical", etc...) that will be interested or influenced by this research; how will this research be used by others; will it be of interest beyond the specific field? The audience for this study is the research studying the Notch signalling pathway. Since dysregulation of this pathway is implicated in a number of devastating diseases, any improved understanding of its mechanistic underpinnings could in the long run lead to better therapeutic management of diseases with significant involvement of malfunctioning Notch signalling.
Please define your field of expertise with a few keywords to help the authors contextualize your point of view. Indicate if there are any parts of the paper that you do not have sufficient expertise to evaluate. Molecular biology, molecular neuroscience, developmental biology, cell-cell signalling, Notch signalling. All parts of the manuscript fall within our expertise.
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Referee #2
Evidence, reproducibility and clarity
Summary
The manuscript describes an analysis of specificity of functional interactions between mammalian Neuralized proteins and different human ligands for Notch. To investigate this, the authors take the approach of constructing hybrid proteins that contain the intracellular domain of the human ligands and the extracellular domain of the Drosophila Delta or Serrate, and investigate their activity in vivo, in the Drosophila wing disc. The latter is a well-established model tissue for assessing Notch ligand activity. As a second assay they express mammalian neutralized constructs in human cells for luciferase-based Notch signal …
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Referee #2
Evidence, reproducibility and clarity
Summary
The manuscript describes an analysis of specificity of functional interactions between mammalian Neuralized proteins and different human ligands for Notch. To investigate this, the authors take the approach of constructing hybrid proteins that contain the intracellular domain of the human ligands and the extracellular domain of the Drosophila Delta or Serrate, and investigate their activity in vivo, in the Drosophila wing disc. The latter is a well-established model tissue for assessing Notch ligand activity. As a second assay they express mammalian neutralized constructs in human cells for luciferase-based Notch signal reporter assays. The experiments are well presented and described and make a strong case for the conclusions that both Neurl1 and 2 can activate Notch signalling by Dll1 and Jag1 but not Dll4 and Jag2. Use of different mutant intracellular domains is used to show the importance of the NXXN motif, which in Drosophila is required for Neuralized interaction with Delta and Serrate. The use of missense mutations and in particular the reactivation of the cryptic NXXD site in Dll4 by substitution to N is convincing for establishing the importance of the motif. There is also colocalization data to support the conclusion that there is likely to be NXXN-dependent complex formation between the ligand and Neuralized proteins. This latter conclusion would be made firmer fi there were pull down data to support it, although to be fair it is most unlikely that another explanation, other than complex formation could account for the observation of both colocalization and ligand activation.
Major comments
The main limitation of the work is that it is mostly based on overexpression of constructs to activate ectopic expression rather than gene editing endogenous genes. It would be helpful if the authors could comment on the limitations of the work in discussion. Two points of data included in the work are important in mitigating this limitation. Firstly, the experiments in the wing disc and cell culture are taking place in a mindbomb mutant background and the activation is observed is therefore a rescue of activity that has been lost. Secondly, and importantly, the final experiment makes use of a Dl mutant Drosophila line which shows embryo lethality when homozygous, with the characteristic neurogenic phenotype. Rescue of lethality can be brought about by knock-in experiments which restore Dl function and this is also true for the ligand hybrid constructs that introduce mammalian ligand intracellular domains only when they include the NXXN motif This indicates the importance of the motif in normal development
Overall, the data presented in the paper is convincing as regards the conclusions made.
Minor points
In figure 1 the legend for D says that cryptic sites are substitutions of N for E or Q, but the figure and main text indicate that the substitutions are N to E or D.
In the remain figures it would be helpful to include in the figure legends and indications of the numbers of wing discs, embryos for which the images shown are representative of.
In Fg 3 The activation of Notch, by neural1 and Dl-Jag1 in B'" is stronger in the ventral side of the disc than the dorsal whereas, although activation of the same ligand by Neurl2 in C'" is weaker the majority of the ectopic wingless expression is on the dorsal compartment. Is there any reason for the switch in preference between the two neutralized proteins? Overgrowth of the wing disc seems to be similar on both sides and so am wondering if the picture is representative of the ectopic wingless distribution in this case.
Significance
Previous work on double genetic knockouts of the two mouse Neuralized genes cast doubt as to whether Neuralized proteins play a role in Notch signal activation in mammals, unlike in Drosophila. There is, however, some genetic indications that spatial memory requires both Notch and neutralized proteins and may represent a specialised function limited to the Neuralized interaction. There are likely to be more subtle contexts waiting to be uncovered. The work is therefore showing important proof of principle for establishing the functionality of the mammalian Neurl proteins and highlights new findings indicting specialisation of the different ligands for interactions with Notch components. Elucidation of such specialisations will help understand why the diversity of different homologues of Notch and ligand have evolved and are maintained in the vertebrate genome compared to the single Notch and two ligands in Drosophila. Since Notch and it misregulation are widely involved in development, health and disease and there is much interest in developing therapeutic interactions that alter Notch activity then the work is likely of broad interest.
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Referee #1
Evidence, reproducibility and clarity
This is an interesting manuscript from two groups of experts in Notch signaling biology with complementary expertise in Drosophila genetics (Klein) and in biophysical studies of the Notch pathway (Sprinzak). The paper provides a cutting-edge structure-function dissection of the E3 ubiquitin ligase Neuralized and its mammalian homologs, Neurl1a and Neurl1a. The work is particularly relevant since the functions of mammalian Neurl1a and Neurl1b have been questioned, and more subtle altogether than those of fly Neuralized (as summarized by the authors in Fig. 1C). This is in part due to the dominant effects of the E3 ubiquitin ligase …
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Referee #1
Evidence, reproducibility and clarity
This is an interesting manuscript from two groups of experts in Notch signaling biology with complementary expertise in Drosophila genetics (Klein) and in biophysical studies of the Notch pathway (Sprinzak). The paper provides a cutting-edge structure-function dissection of the E3 ubiquitin ligase Neuralized and its mammalian homologs, Neurl1a and Neurl1a. The work is particularly relevant since the functions of mammalian Neurl1a and Neurl1b have been questioned, and more subtle altogether than those of fly Neuralized (as summarized by the authors in Fig. 1C). This is in part due to the dominant effects of the E3 ubiquitin ligase Mindbomb1 (Mib1) in Notch ligand-expressing cells from mammalian systems. The authors use careful structure-function work in fly development (mostly wing imaginal discs) and in mammalian cell culture systems, including a clever approach to study the function of mammalian Neurl1a and Neurl1b and mammalian/fly Notch ligand hybrids in Drosophila to draw new conclusions about the function of Neurl1a/b, showing that they can function as activators of Notch signaling mediated by the Notch ligands Dll1 and Jag1, and not by Dll4 and Jag2, tracing these differential effects to the recognition of a short NXXN consensus sequence in the N-terminal region of the ligand's intracellular domain.
Specific questions:
- The current title of the manuscript is not very information-rich and would not allow a reader to gather key information about the findings without reading at least the abstract. Could this be improved? For example, by referring to differential activation of individual Notch ligands, or some other more direct description of the key findings?
- The authors design most key experiments documenting agonistic effects of Neurl1a/1b in a Mib1-deficient background, both in flies and in cell culture systems. This is understandable experimentally to isolate Neurl1a/b's effects in these experimental systems. However, this leaves open questions as to the prevailing effects of Neurl1a/b in cells that also express Mib1 (which the authors comment on in the discussion based on past findings, including some suggesting that Neurl1a/1b can function as Notch inhibitors through a ligand ubiquitination mechanism that may differ from their activating function). Do the authors actually have data that could shed light on this discussion? For example, have they performed cell coculture assays in which Neurl1a or Neurl1b is co-expressed with a Notch ligand, but in the presence of Mib1? This condition seems to be systematically omitted from all the coculture experiments that are presented. It would be interesting to evaluate the net effect of Neurl1a/Neurl1b expression in a Mib1-sufficient system as well.
- The paper suggests important predictions about mammalian functions of Neurl1a/1b, including the neurological effects that have been reported, in double-deficient mice, namely that that there are cells that only express Neurl1a/1b and not Mib1 and do rely on Dll1 and Jag1 for signaling. Could the authors at least comment on this prediction? Are there are any single cell atlases where candidate cells like that can be identified? Or would the authors predict that Neurl1a/1b could actually function as Notch agonist even in cells expressing Mib1? (see also previous comment)
- Some minor typos: line 305 should likely read "flies homozygous for (...)". Line 408, "for providing" repeated twice.
Significance
Thank you for the opportunity to review this lovely collaborative paper. As indicated in my comments to the authors, the findings provide novel structure-function information about an understudied aspect of Notch signaling and clarify conflicting past data about the mammalian homologs of fly Neuralized. The approach is elegant and multidisciplinary, notably in regards to the combination of cell co-culture systems and Drosophila as a platform to study mammalian Neuralized proteins and hybrid Notch ligand molecules. The findings will be interesting to the field and will generate discussion. I would suggest that some additional information would be a plus to substantiate predictions about mammalian functions of Neurl1a/b, and also to clarify its effects in the presence or absence of concomitant Mib1 expression.
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