LGL-1 and the RhoGAP protein PAC-1 redundantly control polarization of the C. elegans embryonic epidermal epithelium

Article activity feed

1. 

   Review Commons

   Dec 18, 2025

   Note: This response was posted by the corresponding author to Review Commons. The content has not been altered except for formatting.

   Learn more at Review Commons

   ---

   Reply to the reviewers

   1. General Statements

   We thank the reviewers for their overall support, thorough review, and thoughtful comments. The points raised were all warranted and we feel that addressing them has improved the quality of our manuscript. Below we respond to each of the points raised.

   2. Point-by-point description of the revisions

   Reviewer #1

   Minor comments:

   Are the lgl-1; pac-1 M-Z- double mutants dead? Only the phenotype of pac-1(M-Z-); lgl-1 (M+Z-) is shown. In figures and text throughout, it should be clear whether mutants are referring to zygotic loss or both maternal and zygotic loss, as this distinction could have major implications on the interpretation …

   Note: This response was posted by the corresponding author to Review Commons. The content has not been altered except for formatting.

   Learn more at Review Commons

   ---

   Reply to the reviewers

   1. General Statements

   We thank the reviewers for their overall support, thorough review, and thoughtful comments. The points raised were all warranted and we feel that addressing them has improved the quality of our manuscript. Below we respond to each of the points raised.

   2. Point-by-point description of the revisions

   Reviewer #1

   Minor comments:

   Are the lgl-1; pac-1 M-Z- double mutants dead? Only the phenotype of pac-1(M-Z-); lgl-1 (M+Z-) is shown. In figures and text throughout, it should be clear whether mutants are referring to zygotic loss or both maternal and zygotic loss, as this distinction could have major implications on the interpretation of experiments.

   Almost all experiments we performed used a combination of RNAi of lgl-1 in a homozygous pac-1 null mutant background, or the other way around. RNAi should eliminate maternal product, but we hesitate to use the terminology M/Z since it has previously been used for protein degradation strategies.

   We have updated the text and figure 1 to address the potential of maternal product masking earlier phenotypes, and performed additional RNAi experiments to demonstrate that the phenotypes obtained by RNAi for either pac-1 or lgl-1 in a homozygous mutant background for the other are the same as for the genetic double mutant. The results are shown as additional images and quantifications in figure 1B,C. We also updated the legend to figure 1 to make it clear that double genetic mutants are obtained from heterozygous lgl-1/+ parents.

   Regarding the phenotype of lgl-1; pac-1 M-Z- double mutants: assuming the reviewer refers to M-Z- double genetic mutants, we cannot make such embryos as the pac-1(M-Z-); lgl-1(M+Z-) animals are already lethal.

   In Figure 1C, it would be more appropriate to show a fully elongated WT embryo to contrast with arrested elongation in mutant embryos.

   We agree with the reviewer and have replaced the 2-fold WT embryo with a 3-fold embryo.

   Is the lateral spread of DLG-1 in double mutant embryos a result of failure to polarize DLG-1, or failure to maintain polarity? This should be straightforward to address in higher time resolution movies.

   We have analyzed additional embryos at early stages of development. In lgl-1; pac-1 embryos we never see the appearance of complete junctions: defects are apparent already at dorsal intercalation. We interpret these results as a failure to properly polarize DLG-1. We have added additional images to Figure S2 and added this sentence to the text: Imaging of embryos from early stages of development on showed that normal continuous junctional DLG-1 bands are never established in pac-1(RNAi); lgl-1(mib201) embryos (Fig. S2B).

   The lack of enhancement of hmp-1(fe4) by lgl-1(RNAi) is quite interesting, given that pac-1 does enhance hmp-1(fe4). To rule out the possibility that this result stems from incomplete lgl-1 RNAi, this experiment should be repeated using the lgl-1 null mutant.

   We have done this experiment by recreating the fe4 S823F mutation in the lgl-1(null) mutant background as well as in the wild-type CGC1 background using CRISPR/Cas9. The phenotype of both was similar, but differs from that of the original PE97 strain. In the original strain, there is ~50% embryonic lethality but worms that complete embryogenesis grow up to be fertile adults. In our new "fe4" strains, nearly all animals are severely malformed with little to no elongation taking place. We are able to maintain both strains (with and without lgl-1) homozygous but with difficulty as only ~5% of animals grow up and give progeny. Apparently, there are genetic differences between PE97 and our CGC1 background that cause phenotypic differences despite having the same amino acid change in HMP-1.

   Nevertheless, using our original embryonic viability criterium of 'hatching', loss of lgl-1 does not enhance the S823F mutation. We have included the following text in the manuscript:

   To rule out that the lack of enhancement by lgl-1(RNAi) is due to incomplete inactivation of lgl-1, we also re-created the hmp-1(fe4) mutation (S823F) by CRISPR in lgl-1(mib201) mutant animals and wild-type controls. The phenotype of the S823F mutant we created is more severe than that of the original PE97 hmp-1(fe4) strain, with only ~5% of animals becoming fertile adults (Fig. S2F). This likely represents the presence of compensatory changes that have accumulated over time in PE97. Nevertheless, consistent with our RNAi results, the presence of lgl-1(mib201) did not further exacerbate the phenotype of HMP-1(S823F) (Fig. S2E, F). Taken together, the lack of enhancement of hmp-1(S823F) mutants by inactivation of loss of lgl-1 This observation argues against a primary role for lgl-1 in regulating cell junctions.

   • Related to point 4, do pac-1 or lgl-1 null mutants enhance partial knockdown of junction protein DLG-1, or is this effect (of pac-1) specific to HMP-1/AJs?*

   We have attempted to address this point using feeding RNAi against dlg-1. However, we were not able to obtain partial depletion of DLG-1. On RNAi feeding plates, control, pac-1, and *lgl-1 *animals did not show significant embryonic lethality. We checked RNAi effectiveness with a DLG-1::mCherry strain and found RNAi by feeding to be very ineffective. Since we could not deplete DLG-1 to a level that results in partial embryonic lethality, we were not able to address this question properly.

   Does lgl-1 loss affect PAC-1 protein localization and vice versa?

   It does not. We have added the following text and a figure panel: Loss-of-function mutants that strongly enhance a phenotype are often interpreted as acting in parallel pathways. We therefore examined whether loss of lgl-1 or pac-1 alters the localization of endogenously GFP-tagged LGL-1 or PAC-1. In neither null background did we detect changes in the subcellular localization of the other protein, consistent with LGL-1 and PAC-1 functioning in parallel pathways (Fig. S1D).

   Reviewer #2

   Very little of the imaging data are analyzed quantitatively, and in many cases it is not clear how many embryos were analyzed. While the images that are presented show clear defects, readers cannot determine how reproducible, strong or significant the phenotypes are.

   We completely agree with the reviewer that interpretation of our data requires this information and apologize for the omission in the first manuscript version. The phenotypes are highly penetrant and consistent (timing of arrest, % lethality, junctional defects), and we have now added quantifications throughout the manuscript.

   In particular, the data below should be quantified and, where possible, analyzed statistically:

   • The frequency of the various junctional phenotypes shown in 2C

   We have now quantified the junctional phenotypes. The junctional defects are highly penetrant: >90% of lgl-1; pac-1 embryos have junctional defects (new Fig. 2B). We used airy-scan confocal imaging to analyze the distribution of the different phenotypes (unaffected, spread laterally, and ring-like pattern). The results are shown in Fig. 2G.

   • The expansion of DLG-1::mCherry in pac-1 lgl-1 embryos should be quantified (related to Figure 2B). For example, the percentage of membrane (marked by PH::GFP) occupied by DLG-1 could be quantified.

   We have performed this quantification, shown in Fig. 2D.

   *- Similarly, the expansion of the aPKC domain should be quantified (Figure *3A).

   An objective quantification of aPKC signal is difficult due to the relatively weak expression of aPKC::GFP and the lack of a clear demarcating boundary. This is part of the reason we measured tortuosity as a more quantifyable indicator of apical domain expansion. We have now added a qualitative observation table as Figure 3B. In addition, we have expanded the quantification of cell geometry by measuring lateral and basal surfaces. Lateral surfaces were decreased. We added the following text:

   To better understand the reason for the change in geometry, we also measured the lengths of the lateral and basal surfaces (Fig. 3F). We found that the absolute lengths of the apical surfaces were not significantly different between pac-1(RNAi); lgl-1(mib201) and control animals. Instead, the lengths of the lateral domain were reduced (Fig. 3F). Hence, the more dome-shaped appearance of epidermal cells in pac-1; lgl-1 double mutant animals is due to a decrease in lateral domain size, which is consistent with the observed lateral spreading of aPKC.

   • *How many embryos were analyzed for each marker shown in Figure 2A, and what proportion showed the described phenotypes? *This could be given in the text or in a panel.

   We have added these numbers to panel 2B, and indicated the percentage in the text.

   • The frequency of the various junctional phenotypes shown in 4F.

   To address this, we have changed figure 4F to show three types of phenotype (strong, mild, no phenotype) and added how frequently we observed each to the panels. In rescue experiments, 18/24 embryos showed no junctional defects, while 6/24 showed a mild defect (compared to 100% severe in non-rescued embryos). To make room for this and other quantifications in Figure 4, we moved the demonstration that PAC-1 is depleted by RNAi to supplemental figure S4.

   Because the genetic perturbations used are global (either deletions or RNAi), it is not established whether PAC-1/LGL-1 act in epidermal epithelial cells per se (versus an earlier requirement that manifests in epidermal epithelial cells). While I agree that this is the most likely scenario, other mechanisms are possible.

   Our experiments indeed use global depletion/deletion of lgl-1 and *pac-1. *We cannot exclude therefore that other tissues do not contribute to the epithelial phenotypes. We assume that other tissues would be affected as well, and in fact have observed abnormal looking pharynx tissue (see our response to reviewer 3 below for examples). As the epidermis is one of the first tissue to develop it is likely the first in which phenotypes become apparent.

   In particular, the overall GFP::aPKC levels appear notably higher in pac-1 lgl-1 embryos in Figure 3A. aPKC levels should be quantified to determine if this is true of pac-1 lgl-1 embryos. If so, couldn't that explain (or at least contribute to) the observed phenotypes?

   Overall higher levels could indeed contribute to the phenotype. However, we have now quantified total aPKC levels in control and pac-1; lgl-1 embryos found no difference between them. We have added the following text to the manuscript: To determine if increased expression of aPKC might explain the broadened apical localization, we measured total intensity levels of aPKC::GFP. However, we detected no differences in fluorescence levels between control and pac-1(RNAi); lgl-1(mib201) animals (Fig. S3B, C).

   Minor

   Figure 4: For completeness, please include the embryonic viability of pac-1 lgl-1 +/- embryos treated with EV and cdc-42(RNAi), as was done for pac-1 lgl-1 pkc-3(ts) in Figure 4E. Presumably the increased proportion of viable embryos with the lgl-1 deletion allele is reflected in an overall increase in embryonic viability.

   The embryonic viability indeed increases, but not as much as one might think because 15% of embryos die from the cdc-42 RNAi itself. The most important rescue argument is that we can obtain adult pac-1; lgl-1 animals with cdc-42 RNAi.

   We have now included the overall rescue and the following text: Overall, cdc-42 RNAi caused a mild increase in embryonic viability (Fig. 4A). However, total embryonic viability may underestimate rescue of pac-1; lgl-1 embryonic lethality, because it also includes the ~15% lethality caused by cdc-42 inactivation itself, even among animals wild type for lgl-1.

   The orientation of the inset images in Figures 2C, 3A and 3D is confusing. An illustration showing how these images are oriented relative to each other would be helpful.

   We have added a figure showing how the junctions are oriented in the figures (Fig. 2E). We have also added supplemental videos S3 and S4 that should illustrate the phenotype more clearly as well.

   For completeness, it would be good to test whether lgl-1(delta) is also synthetically lethal with picc-1(RNAi) (Zilberman 2017).

   We like this idea and had already looked into this. Lgl-1 and picc-1 are not synthetic lethal (see graph in word file submitted). However, PICC-1 is not the only junctional localization signal for PAC-1, as demonstrated by the Nance lab. We find the data interesting but feel that it deserves a more thorough structure/function investigation of PAC-1 than we can provide here. Therefore we would prefer not to include this data.

   Reviewer #3

   We thank the reviewer for their support of our manuscript.

   A few small areas to improve this manuscript:

   p. 6 like 139: "remain" should be "remaining"

   We have fixed this typo.

   Could the authors mention what is the phenotype of the 10% of pac-1 animals that die?

   Yes. They die with pleotropic phenotypes not resembling those of our pac-1; lgl-1 double mutant embryos. We have added examples of these to Figure S1.

   Based on the Supplemental figures, it made me curious to ask: Did the authors notice changes in dorsal epidermal fusions? Cadherin normally disappears in the dorsal hyp7 cells at this time. Did the timing of the fusions change at all?

   We haven't analyzed this in detail but our time-lapse videos show that dorsal fusions still take place and do not seem to be particularly delayed (overall development is slightly delayed but the delay in fusion is consistent with overall delay).

   Again, curiosity driven by the Supplemental figures: did the authors notice defects in apical regions of internal organs, like the pharynx or intestine? The CDC-42 biosensor is asymmetrical in the developing intestine. See: DOI: 10.1242/bio.056911

   We did not pay much attention to the intestine as PAC-1 is barely detectable in this tissue. The pharynx is formed, which we can easily detect in arrested embryos as we use GFP or BFP expressed under the myo-2 promoter to mark the deletion of pac-1. While we did not look closely, we do observe defects in pharynx development.

   Read the original source
2. 

   Review Commons

   Dec 18, 2025

   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

   This manuscript by Jarosinska and colleagues addresses a long-standing mystery in the apical/basal polarity field: why LGL-1 and PAC-1/RhoGAP19D, which are essential in Drosophila, and in some tissue culture contexts, are not essential in C. elegans embryos.

   The authors take an open-ended approach by using genetics, in the form of a genome wide RNAi screen, to find other proteins that enhance the mild phenotypes of lgl-1 mutant embryos. They uncover strong synthetic lethality when they reduce pac-1, a well-documented CDC-42 GAP that supports apical/basal polarity during early embryogenesis, and yet is also only partially required …

   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

   This manuscript by Jarosinska and colleagues addresses a long-standing mystery in the apical/basal polarity field: why LGL-1 and PAC-1/RhoGAP19D, which are essential in Drosophila, and in some tissue culture contexts, are not essential in C. elegans embryos.

   The authors take an open-ended approach by using genetics, in the form of a genome wide RNAi screen, to find other proteins that enhance the mild phenotypes of lgl-1 mutant embryos. They uncover strong synthetic lethality when they reduce pac-1, a well-documented CDC-42 GAP that supports apical/basal polarity during early embryogenesis, and yet is also only partially required during embryogenesis.

   The phenotypic analysis to understand why the embryos die when missing both lgl-1 and pac-1 leads to a careful analysis of known junctional molecules in C. elegans. Using newly made endogenously tagged junctional proteins, including DLG-1 and AFD, so that they can examine all three C. elegans apical junction complexes, the authors find a penetrant defect in the epidermal junctions as the embryos undergo elongation, an actomyosin dependent contractile event that dramatically reshapes the embryos into long, skinny tubes. With disorganized junctions, the embryos die due to ruptures, or hernias, as shown in the Supplemental Movie 2. In addition, and quite excitingly, the apical domains of the embryos are expanded. These defects are then partially rescued by removing CDC-42 or aPKC using RNAi depletion.

   Major comments:

   The claims and conclusions are supported by the data.

   The data is presented in such a way that it is easy to understand what was done, and how measurements were obtained and evaluated.

   Rigorous documentation of how the strains were built and how the genome wide RNAi screen was conducted is included in the Supplemental files.

   Beautiful use of CRISPR to do the genetics:

   since when they made the deletion of lgl-1 they replaced the coding sequence with GFP, they could use GFP to count the animals carrying the deletion in their double mutant analysis with pac-1 deletion mutants.

   Figures are very nicely done.

   The writing is clear.

   Minor comments:

   A few small areas to improve this manuscript:

   p. 6 like 139: "remain" should be "remaining"

   Could the authors mention what is the phenotype of the 10% of pac-1 animals that die?

   Based on the Supplemental figures, it made me curious to ask: Did the authors notice changes in dorsal epidermal fusions? Cadherin normally disappears in the dorsal hyp7 cells at this time. Did the timing of the fusions change at all?

   Again, curiosity driven by the Supplemental figures: did the authors notice defects in apical regions of internal organs, like the pharynx or intestine? The CDC-42 biosensor is asymmetrical in the developing intestine. See: DOI: 10.1242/bio.056911

   Significance

   This study raises interesting and important questions for the general polarity field. Early embryos have hugely redundant methods to maintain apical/basal polarity, which in C. elegans masked the roles for lgl-1 and pac-1 at earlier events, like compaction, when apical/basal polarity is first established. However, during elongation, when healthy strong junctions are a requirement, the double mutant loss of LGL-1 and PAC-1 results in expanded apical domain, that is lethal.

   The study will be of interest to the broader polarity community, and to developmental biologist interested in how the apical junctions are assembled and strengthened during morphogenesis. The Discussion does a good job of showing what aspects of this study are novel, and which support prior findings that suggested, for example, that PAC-1 may have roles independent of CDC-42. I appreciate the comment that our field needs more and more sensitive biosensors to fully address the changes of key polarity regulators.

   Read the original source
3. 

   Review Commons

   Dec 18, 2025

   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 #2

   Evidence, reproducibility and clarity

   Summary: This study focuses on the polarization of epidermal epithelial cells in C. elegans. Whereas the basolateral polarity protein is LGL-1 is required for epithelial polarity in flies, LGL-1 is dispensable for polarization and viability in C. elegans. Through a whole-genome RNAi screen, Jarosinska et al discover that the depletion of the RhoGAP PAC-1 is synthetically lethal with an lgl-1 deletion mutant. pac-1 lgl-1 double mutants have significant polarity defects in the epidermal epithelial, including mislocalization of junctional markers and expansion of the apical aPKC domain. As a result pac-1 lgl-1 double mutants fail to …

   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 #2

   Evidence, reproducibility and clarity

   Summary: This study focuses on the polarization of epidermal epithelial cells in C. elegans. Whereas the basolateral polarity protein is LGL-1 is required for epithelial polarity in flies, LGL-1 is dispensable for polarization and viability in C. elegans. Through a whole-genome RNAi screen, Jarosinska et al discover that the depletion of the RhoGAP PAC-1 is synthetically lethal with an lgl-1 deletion mutant. pac-1 lgl-1 double mutants have significant polarity defects in the epidermal epithelial, including mislocalization of junctional markers and expansion of the apical aPKC domain. As a result pac-1 lgl-1 double mutants fail to maintain surface epithelial and arrest development. Genetic interaction data suggest that increased CDC42 and aPKC activity in pac-1 lgl-1 contributes, as least in part, to the polarity defects and resulting embryonic lethality.

   Major comments:

   Very little of the imaging data are analyzed quantitatively, and in many cases it is not clear how many embryos were analyzed. While the images that are presented show clear defects, readers cannot determine how reproducible, strong or significant the phenotypes are. In particular, the data below should be quantified and, where possible, analyzed statistically:

   • The frequency of the various junctional phenotypes shown in 2C
   • The expansion of DLG-1::mCherry in pac-1 lgl-1 embryos should be quantified(related to Figure 2B). For example, the percentage of membrane (marked by PH::GFP) occupied by DLG-1 could be quantified.
   • Similarly, the expansion of the aPKC domain should be quantified (Figure 3A).
   • How many embryos were analyzed for each marker shown in Figure 2A, and what proportion showed the described phenotypes? This could be given in the text or in a panel.
   • The frequency of the various junctional phenotypes shown in 4F.

   Because the genetic perturbations used are global (either deletions or RNAi), it is not established whether PAC-1/LGL-1 act in epidermal epithelial cells per se (versus an earlier requirement that manifests in epidermal epithelial cells). While I agree that this is the most likely scenario, other mechanisms are possible. In particular, the overall GFP::aPKC levels appear notably higher in pac-1 lgl-1 embryos in Figure 3A. aPKC levels should be quantified to determine if this is true of pac-1 lgl-1 embryos. If so, couldn't that explain (or at least contribute to) the observed phenotypes?

   Minor

   Figure 4: For completeness, please include the embryonic viability of pac-1 lgl-1 +/- embryos treated with EV and cdc-42(RNAi), as was done for pac-1 lgl-1 pkc-3(ts) in Figure 4E. Presumably the increased proportion of viable embryos with the lgl-1 deletion allele is reflected in an overall increase in embryonic viability.

   The orientation of the inset images in Figures 2C, 3A and 3D is confusing. An illustration showing how these images are oriented relative to each other would be helpful.

   For completeness, it would be good to test whether lgl-1(delta) is also synthetically lethal with picc-1(RNAi) (Zilberman 2017).

   Significance

   LGL-1 is a conserved polarity protein that is essential for viability in Drosophila. In contrast, lgl-1 mutants are viable and have weak polarity phenotypes in C. elegans. A previous study showed that LGL-1 acts redundantly with the posterior polarity proteins PAR-2 during establishment of anterior/posterior polarity in the one-cell worm embryo. Here, Jarosinska et al show that LGL-1 acts redundantly with another protein, the RhoGAP protein PAC-1, in the polarization of the embryonic epidermal epithelial. The strength of this study is the identification of redundant roles for PAC-1 and LGL-1, the apparent strength of the polarity defects in the double mutant and the broader implication that LGL-1 may act in a range of redundant, cell/tissue specific pathways to regulate polarity. The primary weakness of this study is the lack of quantification. Additionally, the aPKC and CDC42 genetic interaction data hint at potential pathways, but fall short of establishing LGL-1's or PAC-1's mechanism of action.

   Advance: This works identifies a redundant genetic interaction between LGL-1 and PAC-1. While the data require additional quantification, the phenotypes presented appear clear and strong. Although the molecular mechanism by which LGL-1 and PAC-1 act is not well established in the current work, the core observation is significant and should provide a foundation for future studies dissecting the molecular mechanisms.

   Audience: This work will be of interest to a broad audience. LGL-1 is conserved and its role in cell polarization and epithelial polarity is very actively studied, including in mammalian systems.

   Field of expertise. C elegans embryonic development; cell polarity.

   Read the original source
4. 

   Review Commons

   Dec 18, 2025

   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 #1

   Evidence, reproducibility and clarity

   In this manuscript, Jarosinska and colleagues address the roles of two polarity regulators, pac-1 and lgl-1, in C. elegans epidermal polarity. Loss of function mutations in either of these gene individually does not block polarization, but through a genome-wide RNAi screen, the authors find that pac-1 and lgl-1 enhance each other to cause apical-basal polarity defects and arrest during epidermal morphogenesis. The remainder of the paper focuses on testing genetic interactions between both proteins and AJ proteins (HMP-1) as well as apical proteins (CDC-42, PKC-3). These experiments reveal some interesting differences in how lgl-1 …

   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 #1

   Evidence, reproducibility and clarity

   In this manuscript, Jarosinska and colleagues address the roles of two polarity regulators, pac-1 and lgl-1, in C. elegans epidermal polarity. Loss of function mutations in either of these gene individually does not block polarization, but through a genome-wide RNAi screen, the authors find that pac-1 and lgl-1 enhance each other to cause apical-basal polarity defects and arrest during epidermal morphogenesis. The remainder of the paper focuses on testing genetic interactions between both proteins and AJ proteins (HMP-1) as well as apical proteins (CDC-42, PKC-3). These experiments reveal some interesting differences in how lgl-1 and pac-1 interface with junctional proteins (pac-1 enhances hmp-1 but lgl-1 does not) and apical proteins (lgl-1 suppresses pkc-3 or cdc-42 partial loss but pac-1 does not).

   Minor comments:

   1. Are the lgl-1; pac-1 M-Z- double mutants dead? Only the phenotype of pac-1(M-Z-); lgl-1 (M+Z-) is shown. In figures and text throughout, it should be clear whether mutants are referring to zygotic loss or both maternal and zygotic loss, as this distinction could have major implications on the interpretation of experiments.
   2. In Figure 1C, it would be more appropriate to show a fully elongated WT embryo to contrast with arrested elongation in mutant embryos.
   3. Is the lateral spread of DLG-1 in double mutant embryos a result of failure to polarize DLG-1, or failure to maintain polarity? This should be straightforward to address in higher time resolution movies.
   4. The lack of enhancement of hmp-1(fe4) by lgl-1(RNAi) is quite interesting, given that pac-1 does enhance hmp-1(fe4). To rule out the possibility that this result stems from incomplete lgl-1 RNAi, this experiment should be repeated using the lgl-1 null mutant.
   5. Related to point 4, do pac-1 or lgl-1 null mutants enhance partial knockdown of junction protein DLG-1, or is this effect (of pac-1) specific to HMP-1/AJs?
   6. Does lgl-1 loss affect PAC-1 protein localization and vice versa?

   Significance

   Overall, the manuscript provides additional insights into apical-basal polarization in C. elegans and demonstrates that lgl-1 is likely working in a similar way as in Drosophila, despite the lack of a phenotype in single lgl-1 mutants. I found the experiments to be done rigorously and interpretations of the data appropriate. All of my suggestions on improving the manuscript are minor; suggested experiments should be viewed as optional ways to strengthen the conclusions/impact of the study.

   Read the original source
5. 

   Version published to 10.1101/2025.09.05.674236 on bioRxiv

   Sep 5, 2025