Opposing roles for lipocalins and a CD36 family scavenger receptor in apical extracellular matrix-dependent protection of narrow tube integrity

  1. Alexandra C. Belfi
  2. Sage G. Aviles
  3. Rachel Forman-Rubinsky
  4. Hasreet K. Gill
  5. Jennifer D. Cohen
  6. Aleksandra Nawrocka
  7. Axelle Bourez
  8. Pierre van Antwerpen
  9. Patrick Laurent
  10. Meera V. Sundaram

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Abstract

All exposed epithelial surfaces, including the walls of internal tubes, are lined by a lipid and glycoprotein-rich apical extracellular matrix (aECM) that helps shape and protect the apical domain. Secreted lipocalins are lipid transporters frequently found within apical compartments. We show that loss of the C. elegans lipocalin LPR-1 disrupts the assembly of another lipocalin, LPR-3, within the pre-cuticle aECM that protects and shapes the narrow excretory duct and pore tubes. LPR-1 is apically secreted and colocalizes with LPR-3 in intracellular vesicles and lysosomes, but unlike LPR-3 it does not detectably incorporate into the aECM. Forward genetic screens for lpr-1 suppressors identified mutations in scav-2, which encodes a transmembrane protein of the CD36 scavenger receptor B family. Loss of scav-2 restored LPR-3 matrix localization and suppressed the lpr-1 tube shaping defect, as well as the tube-shaping defects of a subset of pre-cuticle mutants, but not lpr-3 mutants. A SCAV-2 fusion accumulated at apical surfaces of interfacial epithelial tubes, including the excretory duct and pore, and both tissue-specific suppression of lpr-1 matrix defects and tissue-specific rescue experiments support a local role for SCAV-2 within these tubes. These data demonstrate that LPR-1 and SCAV-2 have opposing effects on narrow tube integrity by altering the content and organization of that tube’s luminal aECM, possibly by acting as transporters of an LPR-3 cofactor. These results have broadly relevant implications regarding the importance of lipocalins and scavenger receptors for aECM organization and integrity of the narrowest tubes in the body.

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    Reply to the Reviewers

    We thank the reviewers for their positive assessments overall and for many helpful suggestions for clarification to make the manuscript more accessible to a broader audience. We made minor text changes and added more labels to the figures to address these comments.

    __Referee #1

    __

    Summary: In this study, the authors show a genetic interaction of the lipid receptors Lpr-1, Lpr-3 and Scav-2 in C. elegans. They show that Lpr-1 loss-of-function specifically affects aECM localization of Lpr-3 and attribute the lethality of Lpr-1 mutants to this phenotype. The authors performed a mutagenesis screen and identified a third lipid receptor, Scav-2, as a modulating factor: loss of scav-2 partially rescues the Lpr-1 phenotype. The authors created a variety of tools for this study, notably Crispr-Cas9-mediated knock-ins for endogenous tagging of the receptors.

    *Major comments: *

    while the authors provide a nice diagram showing the potential roles and interplay of lpr-1, lpr-3 and scav-2, it remains unclear what their respective cargo is. The nature of interaction between the proteins remains unclear from the data.

    Response

    • We agree that identifying the relevant cargo(s) will be key to understanding the detailed mechanisms involved and that the lack of such information is a limitation of our study. However, the impact of our study is to show that these lipid transporters functionally interact to affect aECM organization, a role that could be relevant to many systems, including humans.

    As an optional (since time-consuming) experiment I would suggest trying more tissue-specific lipidomics.

    Response

    • This would be an interesting future experiment but is outside our current technical capabilities.

    The lipidomics data should be presented in the figures, even if there were no significant changes. Importantly, show the lipid abundance at least of total lipids, better of individual classes, normalized to the material input (e.g. number of embryos, protein).

    Response

    • The reviewer is right to point out that lipid variations could occur at different levels, and that we should exercise caution. However, the unsupervised lipidomics analysis would have detected not only individual lipid variations, but also variations in the total or subgroup lipid content. Indeed, the eggs were weighed prior to extraction and each sample was extracted with the same precise volume of solvent before analysis. Furthermore, the LC-MS/MS injection sequence included blanks and quality control (QC) samples. The blanks were the extraction solvent, which allowed us to control for features unrelated to the biological samples. The QC sample was a mixture of all the samples included in the injection sequence, reflecting the central values of the model. If a subclass of samples, such as the lpr-1 mutant, had been characterized by a decrease in one lipid, a subgroup of lipids, or all lipids, it would have clustered separately. Instead, our PCA showed that the variation between samples of the same genotype (wild type, lpr-1 mutant, or lpr-1; scav-2) was similar to the variation between samples from two different genotypes. This means that we did not detect modifications to lipid quantity specifically or in total. A figure illustrating the lipid contents would show no difference between groups.

    Figure 1g: I do not understand what the lpr3:gfp signal is: the punctae in the overview image? and where are they in the zoom image showing anulli and alae? Also, how where the anulli and alae structures labeled? please provide more information

    Response

    • All of the fluorescent signal shown in this figure panel corresponds to the indicated LPR fusion - no other labelling method was used. SfGFP::LPR-3 labels the matrix structures (alae and annuli) as well as some puncta – the ratio of matrix to puncta changes over developmental stages. We edited the figure legend to make this more clear.

    One point that is not sufficiently adressed is that the authors deduce from the inability of the scav-2 gfp knock in to suppress lpr1 lethality that scav2 function is not impaired. This is quite indirect. Can the authors provide more convincing evidence that scav-2 ki has normal function?

    Response

    • Suppression of *lpr-1 *(or other aECM mutant) lethality is the only known phenotype caused by loss of scav-2 Therefore, this is the only phenotype for which we can do a rescue experiment to test functionality of the knock-in. The data presented do indicate that the knock-in fusion retains significant function.

    In general, the data is clearly presented and the statistical analyses look sound.

    Response

    • Thank you

    __Minor comments: __

    Please provide page and line numbers!

    Response:

    • done

    Avoid contractions like "don't" in both text and figure legends

    Response:

    • changed one instance of “don’t” to “do not”

    Page 12: I do not understand the meaning of the sentence "This transgene also caused more modest lethality in a wild-type background"

    Response:

    • Wording changed to “This transgene caused very little lethality in a wild-type background (Fig. 6C), indicating it is not generally toxic.”

    Figure 7: what is meant with "Dodt"?

    Response:

    • Dodt gradient contrast imaging is a method for transmitted light imaging similar to DIC and is used on some confocal microscopes. It is now explained in the Methods section. We removed the Dodt label from Figure 7 since it seems to be confusing and it is not really important whether the brightfield image is DIC or Dodt.

    Reviewer #1 (Significance (Required)):

    The study is experimentally sound and uses numerous novel tools, such as endogenously tagged lipid receptors. It is an interesting study for researchers in basic research studying lipid receptors and ECM biology. It provides insights on the genetic interaction of lipid receptors. My expertise is in lipid biochemistry, inter-organ lipid trafficking and imaging. I am not very familiar with C. elegans genetics.

    __Referee #2 __

    1. The manuscript is very well written; the documentation is fine, but some more details are needed for better following the subject for readers not familiar with nematode anatomy.

    For instance, while alae are somehow explained, annuli are not - structures that look abnormal in lpr1 and lpr1-scav2 mutants (Fig. 5B).

    Response

    • Apologies for this oversight. We added annuli labels to Figure 1 and Figure 5 panels and added descriptions of annuli to the Figure 1 legend and the Results text.

    Moreover, the authors show in Fig. 1 the punctae etc in the epidermis, whereas in Fig. 2 the show Lpr3 accumulation or not in the duct and the pore (lpr1). How do they localize in the cells of these structures at high magnification? It is also important to see the Lpr3 localisation in lpr1 mutants shown in Fig. 2A with the quality of the images shown in Fig. 1F. This applies also to Figs. 4 and 5.

    Responses:

    • The embryonic duct and pore cells are very small and we have not reliably seen puncta within them. In Figs 2 and 5, we supplemented the duct and pore images with those from the epidermis, which is a much larger tissue, allowing us to resolve puncta and matrix structures with better resolution.
    • The laser settings in Figs 2,4,5 (as opposed to Fig. 1) were chosen to avoid saturation of the matrix signal so that we could do accurate quantifications as shown. The images are unmodified with respect to brightness and therefore appear relatively dim – but we think they convey the observations very accurately.

    I would like to see punctae in lpr1-scav2 doubles.

    Response:

    • Puncta in this genotype are shown for the epidermis in Figure 5. It has not been possible to see puncta specifically within the embryonic duct and pore.

    Regarding the central mechanism, one possibility is - what the authors describe - that Lpr1 is needed for Lpr3 accumulation in ducts and tubes. Alternatively, Lpr1 is needed for duct and tube expansion, in lack of which Lpr3 is unable to reach its destination that is the lumina. Scav2, in this scenario, might be antagonist of tube and duct expansion, and thereby rescue the Lpr1 mutant phenotype independently. Admittedly, the non-accumulation of Lpr3 in scav2 mutants argues against a lpr1-independent function of scav2.

    Responses:

    • LPR-1 is indeed needed to maintain duct and pore tube integrity as the tubes grow, but in mutants the tubes appear to collapse at a later stage than we imaged here (Stone et al 2009). The ~normal accumulation of LET-4 and LET-653 further argues that the duct and pore tubes are still intact at the 1.5-to-2-fold stages. Therefore, we conclude that the defect in LPR-3 accumulation precedes duct and pore collapse.
    • The changes we document in the epidermis also show that the lpr-1 mutant affects LPR-3 accumulation in another (non-tube) tissue.

    In any case, to underline the aspect of Lpr1-Scav2 dosage relationship, the authors may also have a look at Lpr3 distribution in lpr1 heterozygous, and lpr1-scav2 double heterozygous worms. In this spirit, it would be interesting to see the semi-dominant effects of scav2 on Lpr3 localisation in lpr1 mutants by microscopy.

    Response:

    • Because of the hermaphroditism of C. elegans, it would be technically challenging to confidently identify heterozygous (vs. homozygous) embryos for confocal imaging. We do not think that the results would be informative enough to warrant the effort, given that we’ve already shown that scav-2 heterozygosity can partly suppress lpr-1 The expectation is that LPR-3 levels would be partially restored in the scav-2 het, but it might take a very large sample size to confidently assess that partial effect.

    One word to the overexpression studies: it is surprising that the amounts of Scav2 delivered by the expression through the grl-2 promoter in the lpr1, scav2 background are almost matching those by the opposite effect of scav2 mutations on lpr1 dysfunction.

    Response:

    • The reviewer refers to the transgenic rescue experiment with the grl-2pro::SCAV-2 transgene. Because the scav-2 mutant phenotype being tested is suppression of lpr-1 lethality, the expected result from scav-2 rescue is to restore the lpr-1 lethal phenotype to the strain. This is exactly the result we see. We have revised the text to more clearly explain the logic.

    One issue concerns the localization of scav2-gfp "rarely" in vesicles: what are these vesicles?

    Response

    • Only a handful of vesicles were seen across all the images we collected, and we have not yet identified them. They could be associated with either SCAV-2 delivery or removal from the plasma membrane, as now stated in the text. SCAV-2 trafficking would be an interesting area for further study but is beyond the scope of this paper.

    One comment to the Let653 transgenes/knock-ins: the localization of transgenic Let653-gfp may be normal in lpr1 mutants because there are wild-type copies in the background.

    Response

    • There are wild type copies of LET-653 in the background, but no wild type copies of LPR-1. Even if the untagged LET-653 would be recruiting the tagged LET-653 as the reviewer suggests, we can still conclude that lpr-1 loss does not prevent the untagged LET-653 (and thus also the tagged LET-653) from accumulating in the duct lumen matrix.

    One thought to the model: if Scav2 has a function in a lpr1 background, this means that yet another transporter X delivers the substrate for Scav2, isn't it?

    Response

    • Yes, we completely agree with this interpretation and have revised the discussion and Figure 8 legend to more explicitly make this point.

    A word to the term haploinsifficient that is used in this study: scav2 mutants would be haploinsifficient if the heterozygous worms died in an otherwise wild-type background.

    Response

    • We disagree with this comment. The term “haploinsufficient” simply means that heterozygosity for a deletion or other loss of function allele can cause a mutant phenotype – the term is not restricted to lethal phenotypes.

    Reviewer #2 (Significance (Required)):

    Alexandra C.Belfi and colleagues wrote the manuscript entitled "Opposing roles for lipocalins and a CD36 family scavenger receptor in apical extracellular matrix-dependent protection of narrow tube integrity" in which they report on their findings on the genetic and cell-biological interaction between the lipid transporters Lpr1 and scav2 in the nematode C. elegans. In principle, these two proteins are involved in shaping the apical extracellular matrix (aECM) of ducts by regulating the amounts of Lpr3 in the extracellular space. While seems to act cell autonomously, Lpr1 has a non-cell autonomous effect on Lpr3.

    __Referee #3 __ Summary: Using a powerful combination of genetic and quantitative imaging approaches, Belfi et al., describe novel findings on the roles of several lipocalins-secreted lipid carrier proteins-in the production and organization of the apical extracellular matrix (aECM) required for small diameter tube formation in C. elegans. The work comprises a substantial extension of previous studies carried out by the Sundaram lab, which has pioneered studies into the roles of aECM and accessory proteins in creating the duct-pore excretion tube and which also plays a role in patterning of the epidermal cuticle. One core finding is that the lipocalin LPR-1 does not stably associate with the aECM but is instead required for the incorporation of another lipocalin, LPR-3. A second major finding is that reduction of function in SCAV-2, a SCARB family membrane lipid transporter, suppresses lpr-1 mutant lethality along with associated duct-pore defects and mislocalization of LPR-3. Likewise loss of scav-2 partially suppresses defects in two other aECM proteins and restores defects in LPR-3 localization in one of them (let-653). Additional genetic and protein localization studies lead to the model that LPR-1 and SCAV-2 may antagonistically regulate one or more lipid or lipoprotein factors necessary for LPR-3 localization and duct-pore formation. A role for LPR-1 and LPR-3 at lysosomes is clearly implicated based on co-localization studies, although a specific role for lysosomes (or related organelles) is not defined. Finally, MS data suggests that neither LPR-1 or SCAV-2 grossly affect lipid composition in embryos, consistent with dietary interventions failing to affect mutant phenotypes. Ultimately, a plausible schematic model is presented to explain for much of the data.

    __*Major comments:

    *__

    The studies are very thorough, convincing, and generally well described. Conclusions are logical and well grounded. Additional experiments are not required to support the authors major conclusions, and the data and methods are described in a sufficient detail to allow replication. As such my comments are minor and should be addressable at the author's discretion in writing.

    Response

    • Thank you for these positive comments

    __*Minor comments: *__2) In the abstract, "tissue-specific suppression" made me think that there was going to be a tissue-specific knockdown experiment, which was not the case. Rather scav-2 suppression is specific to the duct-pore, which corresponds to where scav-2 is expressed. Consider rewording this.

    Response

    • Wording was changed to “duct/pore-specific suppression”
    1. Page 5. Suggest wording change to, "Whereas LPR-3 incorporates stably into the precuticle, suggesting a structural role in matrix organization, LPR-1..."

    Response

    • Done
    1. LIMP-2 versus LIMP2. Both are used. Uniprot lists LIMP2, but some papers use LIMP-2. Choose one and be consistent.

    Response

    • Everything changed to LIMP2.
    1. Some of the data for S6 Fig wasn't referred to directly in the text. Namely results regarding pcyt-1 and pld-1. I'd suggest incorporating this into the results section possibly using, "As a control for our lipid supplementation experiments..."

    Response

    • These experiments are now described on page 11.
    1. Page 12 bottom. I understand the use of "oppose", but another way to put it is that SCAV-2 and LPR-1 (antagonistically or collectively) modulate aECM composition. Other terms that might confuse some readers is the use of upstream and downstream, although I OK with its use in the context of this work.

    Response

    • The genetics indicate that lpr-1 and scav-2 have opposite effects on tube shaping and LPR-3 localization, so they do function antagonistically rather than collectively/cooperatively; we decided to keep this terminology.
    1. Page 16. I understand the logic that SCAV-2 is unlikely to directly modulate LPR-3 given its presumed molecular function. But is it possible that LPR-3 levels are already maxed out in the aECM so that loss of SCAV-2 doesn't lead to any increase? Conversely, one could argue that even if acting indirectly, SCAV-2 could have led to increased LPR-3 levels, unless they were already maxed.

    Response

    • This is a good point and the possibility is now mentioned in the Results page 9. We also changed our wording in the Abstract and Discussion to acknowledge the possibility that LPR-3 could be the SCAV-2 cargo, though we still don’t favor this model.
    1. Figure legend 1. I did not see an asterisk in figure 1B.

    Response

    • thanks for catching this error, text removed
    1. Figure 1C. Might want to define the "degree" term in the legend for people outside the field.

    Response

    • We added an explanation to the figure legend.
    1. Fig 1 G. I was just wondering if cuticle autofluorescence was an issue for taking these images.

    Response

    • Cuticle auto fluorescence is generally quite dim in L4s with our settings, and it was not an issue at this mid/late L4 stage, which corresponds to when both LPR fusions are at their brightest. Note that both large panels are MAX projections and yet you can’t see any cuticle auto-fluorescence in the LPR-1 panel.
    1. Fig 2 and others. Please define error bars.

    Response

    • These correspond to the standard deviation; this information is now added to the Methods.
    1. Fig 5. From the images, it looks like lpr-1; scav-2 doubles might have a worse (pre)cuticle defect in LPR-3 localization than lpr-1 singles. If so that would be interesting and would suggest that their relationship with respect to the modulation of LPR-3 is context dependent. Admittedly, the lack of obvious scav-2 expression in the epidermis would not be consistent with an effect (positive or negative).

    Response

    • The lpr-1 scav-2 strain is certainly not improved over lpr-1 but we have not noted any consistent worsening of the phenotype either.
    1. Consider defining Dodt in the first figure legend where it appears.

    Response

    • Dodt gradient contrast imaging is a method of transmitted light imaging similar to DIC and is used on some confocal microscopes. It is now explained in the Methods section. We removed the term from Figure 7 since it seems to be confusing.
    1. For Mander's, is there a reason to report just one of the two findings (M1 or M2) versus both?

    Response

    • We now include the 2nd Manders value in the figure legend and note that value is much lower (0.25) because much of the red signal is lysosomes (where green would be quenched by acidity).
    1. Consider referring to specific panels (A, B...) within references to the supplemental files.

    Response

    • done
    1. Fig S6E. Neither "increasing nor increasing" to "increasing nor decreasing".

    Response

    • fixed

    **Referees cross-commenting**

    I thought that Reviewers 1 and 2 brought up some good points. My sense is that Belfi and colleagues can address most of these in writing, but are of course welcome to add new data as they see fit. I get that it's not a "perfect" paper where everything is explained fully or comes together, but I don't see that as a flaw that needs to be fixed. I think that the manuscript represents a good deal of work (as it is) and provides a sufficient advance while also suggesting an interesting link to disease. It will be up to individual journals to decide if the findings meets their criteria.

    Reviewer #3 (Significance (Required)):

    Significance: The work carried out in this paper, and more generally by the Sundaram lab, always has a ground-breaking element because very few labs in the field have studied in detail the developmental roles and regulation of the aECM, in large part because it can be challenging to dissect. The core findings in this study are rather novel and unexpected, namely the opposing roles of the paralogous LPR-1 and LPR-3 lipocalins and their functional interactions with SCAV-2. The study does stop short of finding specific molecules (lipid or lipoprotein) that would mediate the effects they report, and it wasn't yet clear how the lysosomal co-loc plays a role, but this is not a criticism of the work presented or the forward progress. I was particularly intrigued by the idea, presented in the discussion, that disruption of vascular aECM could potentially account for some of the (complex) observations regarding the role of lipocalins and SCARB proteins in human disease. This would represent a new avenue for researchers to consider and underscores the power of using non-biased approaches in model systems.

    As for all my reviews, this is signed by David Fay.

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    Referee #3

    Evidence, reproducibility and clarity

    Summary:

    Using a powerful combination of genetic and quantitative imaging approaches, Belfi et al., describe novel findings on the roles of several lipocalins-secreted lipid carrier proteins-in the production and organization of the apical extracellular matrix (aECM) required for small diameter tube formation in C. elegans. The work comprises a substantial extension of previous studies carried out by the Sundaram lab, which has pioneered studies into the roles of aECM and accessory proteins in creating the duct-pore excretion tube and which also plays a role in patterning of the epidermal cuticle. One core finding is that the lipocalin LPR-1 does not stably associate with the aECM but is instead required for the incorporation of another lipocalin, LPR-3. A second major finding is that reduction of function in SCAV-2, a SCARB family membrane lipid transporter, suppresses lpr-1 mutant lethality along with associated duct-pore defects and mislocalization of LPR-3. Likewise loss of scav-2 partially suppresses defects in two other aECM proteins and restores defects in LPR-3 localization in one of them (let-653). Additional genetic and protein localization studies lead to the model that LPR-1 and SCAV-2 may antagonistically regulate one or more lipid or lipoprotein factors necessary for LPR-3 localization and duct-pore formation. A role for LPR-1 and LPR-3 at lysosomes is clearly implicated based on co-localization studies, although a specific role for lysosomes (or related organelles) is not defined. Finally, MS data suggests that neither LPR-1 or SCAV-2 grossly affect lipid composition in embryos, consistent with dietary interventions failing to affect mutant phenotypes. Ultimately, a plausible schematic model is presented to explain for much of the data.

    Major comments:

    The studies are very thorough, convincing, and generally well described. Conclusions are logical and well grounded. Additional experiments are not required to support the authors major conclusions, and the data and methods are described in a sufficient detail to allow replication. As such my comments are minor and should be addressable at the author's discretion in writing.

    Minor comments:

    1. In the abstract, "tissue-specific suppression" made me think that there was going to be a tissue-specific knockdown experiment, which was not the case. Rather scav-2 suppression is specific to the duct-pore, which corresponds to where scav-2 is expressed. Consider rewording this.

    2. Page 5. Suggest wording change to, "Whereas LPR-3 incorporates stably into the precuticle, suggesting a structural role in matrix organization, LPR-1..."

    3. LIMP-2 versus LIMP2. Both are used. Uniprot lists LIMP2, but some papers use LIMP-2. Choose one and be consistent.

    4. Some of the data for S6 Fig wasn't referred to directly in the text. Namely results regarding pcyt-1 and pld-1. I'd suggest incorporating this into the results section possibly using, "As a control for our lipid supplementation experiments..."

    5. Page 12 bottom. I understand the use of "oppose", but another way to put it is that SCAV-2 and LPR-1 (antagonistically or collectively) modulate aECM composition. Other terms that might confuse some readers is the use of upstream and downstream, although I OK with its use in the context of this work.

    6. Page 16. I understand the logic that SCAV-2 is unlikely to directly modulate LPR-3 given its presumed molecular function. But is it possible that LPR-3 levels are already maxed out in the aECM so that loss of SCAV-2 doesn't lead to any increase? Conversely, one could argue that even if acting indirectly, SCAV-2 could have led to increased LPR-3 levels, unless they were already maxed.

    7. Figure legend 1. I did not see an asterisk in figure 1B.

    8. Figure 1C. Might want to define the "degree" term in the legend for people outside the field.

    9. Fig 1 G. I was just wondering if cuticle autofluorescence was an issue for taking these images.

    10. Fig 2 and others. Please define error bars.

    11. Fig 5. From the images, it looks like lpr-1; scav-2 doubles might have a worse (pre)cuticle defect in LPR-3 localization than lpr-1 singles. If so that would be interesting and would suggest that their relationship with respect to the modulation of LPR-3 is context dependent. Admittedly, the lack of obvious scav-2 expression in the epidermis would not be consistent with an effect (positive or negative).

    12. Consider defining Dodt in the first figure legend where it appears.

    13. For Mander's, is there a reason to report just one of the two findings (M1 or M2) versus both?

    14. Consider referring to specific panels (A, B...) within references to the supplemental files.

    15. Fig S6E. Neither "increasing nor increasing" to "increasing nor decreasing".

    As for all my reviews, this is signed by David Fay.

    Referees cross-commenting

    I thought that Reviewers 1 and 2 brought up some good points. My sense is that Belfi and colleagues can address most of these in writing, but are of course welcome to add new data as they see fit. I get that it's not a "perfect" paper where everything is explained fully or comes together, but I don't see that as a flaw that needs to be fixed. I think that the manuscript represents a good deal of work (as it is) and provides a sufficient advance while also suggesting an interesting link to disease. It will be up to individual journals to decide if the findings meets their criteria.

    Significance

    Significance:

    The work carried out in this paper, and more generally by the Sundaram lab, always has a ground-breaking element because very few labs in the field have studied in detail the developmental roles and regulation of the aECM, in large part because it can be challenging to dissect. The core findings in this study are rather novel and unexpected, namely the opposing roles of the paralogous LPR-1 and LPR-3 lipocalins and their functional interactions with SCAV-2. The study does stop short of finding specific molecules (lipid or lipoprotein) that would mediate the effects they report, and it wasn't yet clear how the lysosomal co-loc plays a role, but this is not a criticism of the work presented or the forward progress. I was particularly intrigued by the idea, presented in the discussion, that disruption of vascular aECM could potentially account for some of the (complex) observations regarding the role of lipocalins and SCARB proteins in human disease. This would represent a new avenue for researchers to consider and underscores the power of using non-biased approaches in model systems.

  3. Review Commons

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    Referee #2

    Evidence, reproducibility and clarity

    The manuscript is very well written; the documentation is fine, but some more details are needed for better following the subject for readers not familiar with nematode anatomy. For instance, while alae are somehow explained, annuli are not - structures that look abnormal in lpr1 and lpr1-scav2 mutants (Fig. 5B). Moreover, the authors show in Fig. 1 the punctae etc in the epidermis, whereas in Fig. 2 the show Lpr3 accumulation or not in the duct and the pore (lpr1). How do they localize in the cells of these structures at high magnification? It is also important to see the Lpr3 localisation in lpr1 mutants shown in Fig. 2A with the quality of the images shown in Fig. 1F. This applies also to Figs. 4 and 5. I would like to see punctae in lpr1-scav2 doubles. Regarding the central mechanism, one possibility is - what the authors describe - that Lpr1 is needed for Lpr3 accumulation in ducts and tubes. Alternatively, Lpr1 is needed for duct and tube expansion, in lack of which Lpr3 is unable to reach its destination that is the lumina. Scav2, in this scenario, might be antagonist of tube and duct expansion, and thereby rescue the Lpr1 mutant phenotype independently. Admittedly, the non-accumulation of Lpr3 in scav2 mutants argues against a lpr1-independent function of scav2. In any case, to underline the aspect of Lpr1-Scav2 dosage relationship, the authors may also have a look at Lpr3 distribution in lpr1 heterozygous, and lpr1-scav2 double heterozygous worms. In this spirit, it would be interesting to see the semi-dominant effects of scav2 on Lpr3 localisation in lpr1 mutants by microscopy. One word to the overexpression studies: it is surprising that the amounts of Scav2 delivered by the expression through the grl-2 promoter in the lpr1, scav2 background are almost matching those by the opposite effect of scav2 mutations on lpr1 dysfunction.

    One issue concerns the localization of scav2-gfp "rarely" in vesicles: what are these vesicles?

    One comment to the Let653 transgenes/knock-ins: the localization of transgenic Let653-gfp may be normal in lpr1 mutants because there are wild-type copies in the background.

    One thought to the model: if Scav2 has a function in a lpr1 background, this means that yet another transporter X delivers the substrate for Scav2, isn't it?

    A word to the term haploinsifficient that is used in this study: scav2 mutants would be haploinsifficient if the heterozygous worms died in an otherwise wild-type background.

    Significance

    Alexandra C.Belfi and colleagues wrote the manuscript entitled "Opposing roles for lipocalins and a CD36 family scavenger receptor in apical extracellular matrix-dependent protection of narrow tube integrity" in which they report on their findings on the genetic and cell-biological interaction between the lipid transporters Lpr1 and scav2 in the nematode C. elegans. In principle, these two proteins are involved in shaping the apical extracellular matrix (aECM) of ducts by regulating the amounts of Lpr3 in the extracellular space. While seems to act cell autonomously, Lpr1 has a non-cell autonomous effect on Lpr3.

  4. Review Commons

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    Referee #1

    Evidence, reproducibility and clarity

    Summary: In this study, the authors show a genetic interaction of the lipid receptors Lpr-1, Lpr-3 and Scav-2 in C. elegans. They show that Lpr-1 loss-of-function specifically affects aECM localization of Lpr-3 and attribute the lethality of Lpr-1 mutants to this phenotype. The authors performed a mutagenesis screen and identified a third lipid receptor, Scav-2, as a modulating factor: loss of scav-2 partially rescues the Lpr-1 phenotype. The authors created a variety of tools for this study, notably Crispr-Cas9-mediated knock-ins for endogenous tagging of the receptors.

    Major comments: while the authors provide a nice diagram showing the potential roles and interplay of lpr-1, lpr-3 and scav-2, it remains unclear what their respective cargo is. The nature of interaction between the proteins remains unclear from the data. As an optional (since time-consuming) experiment I would suggest trying more tissue-specific lipidomics. The lipidomics data should be presented in the figures, even if there were no significant changes. Importantly, show the lipid abundance at least of total lipids, better of individual classes, normalized to the material input (e.g. number of embryos, protein). Figure 1g: I do not understand what the lpr3:gfp signal is: the punctae in the overview image? and where are they in the zoom image showing anulli and alae? Also, how where the anulli and alae structures labeled? please provide more information One point that is not sufficiently adressed is that the authors deduce from the inability of the scav-2 gfp knock in to suppress lpr1 lethality that scav2 function is not impaired. This is quite indirect. Can the authors provide more convincing evidence that scav-2 ki has normal function? In general, the data is clearly presented and the statistical analyses look sound.

    Minor comments: Please provide page and line numbers! Avoid contractions like "don't" in both text and figure legends Page 12: I do not understand the meaning of the sentence "This transgene also caused more modest lethality in a wild-type background" Figure 7: what is meant with "Dodt"?

    Significance

    The study is experimentally sound and uses numerous novel tools, such as endogenously tagged lipid receptors. It is an interesting study for researchers in basic research studying lipid receptors and ECM biology. It provides insights on the genetic interaction of lipid receptors.

    My expertise is in lipid biochemistry, inter-organ lipid trafficking and imaging. I am not very familiar with C. elegans genetics.

  5. Version published to 10.1101/2025.07.25.666821 on bioRxiv