IFT20 regulates lymphatic endothelial cell-cell junctions via endocytic trafficking of VE-cadherin

  1. Delayna Paulson
  2. Ahana Majumder
  3. Zachary Lehmann
  4. Luke Knutson
  5. Jacob Paulson
  6. Shannon Lasey
  7. Darci M. Fink

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Abstract

Intraflagellar transport (IFT) proteins are required for the assembly and function of primary cilia. They also regulate non-ciliary polarized vesicular traffic, such as T cell receptor recycling. We recently reported that lymphatic endothelial cells assemble primary cilia and express IFT proteins. Here, we report that IFT20 regulates vascular endothelial cadherin (VE-cadherin) localization at adherens junctions. IFT20 deletion caused discontinuous, button-like interendothelial junctions. This resulted in excessive lymphangiogenesis and impaired lymph drainage in mice. In vitro, VEGF-C treatment of IFT20 KD primary human dermal lymphatic endothelial cells caused accumulation of VE-cadherin in RAB5+ endosomes and enhanced and sustained VEGFR-3 signaling. Our findings are consistent with a model in which IFT20 promotes recycling of VE-cadherin to the adherens junction where it sequesters VEGFR-3 at the cell surface, thereby limiting pro-lymphangiogenic signaling. In the absence of IFT20, intercellular junctions are destabilized, pro-lymphangiogenic VEGFR-3 signaling is enhanced, and lymph transport is impaired by intracellular sequestration of VE-cadherin. This study elucidates the function of an IFT protein in lymphatic endothelial cells and provides mechanistic insight into the processes that regulate lymphatic endothelial cell-cell junctions and lymphangiogenic signaling.

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    __Reviewer #1 __

    (Evidence, reproducibility and clarity (Required)):

    The manuscript identified a novel role of Intraflagellar Transport Protein 20 (IFT20) in the function and homeostasis of lymphatic endothelial junctions. The authors showed that IFT20 regulates VE-cadherin localization at adherens junctions in lymphatic endothelial cells. The authors performed impressive in vivo work that shows the requirement for IFT20 for the homeostasis of intercellular junctions, lymphangiogenesis, and drainage function of lymphatic vessels. In contrast, the cell biology part of the paper was underwhelming and will need significant revisions to support the proposed model. In the result section, several conclusions have to be toned down to match the actual results. The study employs in vivo mouse models, immunofluorescence, biochemical assays, and loss-of-function experiments to support their conclusions.

    Major comments

    • The authors present disrupted localization of VE-cadherin. Is this a mislocalization and/or protein stability issue in IFT20 KD cells? A western blot can help assess protein levels, and a phase-chase endocytosis assay of VE-cadherin can strengthen evidence. The authors did not confirm the permeability phenotype seen in vivo.

    We thank the reviewer for this helpful suggestion.

    Planned 1: Western blot to assess total VE-cadherin protein levels in IFT20 WT and KD cells.

    Planned 2: Immunofluorescence staining for cell-surface VE-cadherin using permeabilized and non-permeabilized IFT20 WT and KD cells during VEGF-C stimulation and washout.

    Together, these two experiments will assess VE-cadherin stability and more directly test the hypothesis that VE-cadherin does not recycle effectively back to the cell surface in the absence of IFT20.

    • While the authors focused on IFT20 and rab5, we do not have a clear idea about the vesicular dynamics as well as the status of early, late, and recycling endosomes in IFT20 KD cells. Is IFT 20 localized to non-rab5+ endosomes, and if yes, what are the species? A more general endosomal profiling would help strengthen the authors' message. For example, in Fig. 4-5, the authors will have to stain for other early endosomal markers as well as late, and recycling endosomal markers in control and IFT20 KD cells.

    Thank you for this helpful suggestion.

    Planned 3: Immunofluorescence staining for EEA1 (early endosome), RAB7 (late endosome), RAB4 (fast recycling), RAB11 (recycling endosome) along with IFT20 to determine its localization pattern.

    This experiment will determine the localization of IFT20 relative to various endosomal compartments.

    • In fig. 6C, a majority of VE-cadherin is not associated with Rab5. Staining with additional endosomal markers might help identify other endosomal species colocalizing with VE-cadherin. It will be critical to add to Fig. 6c the intensity profiles depicting colocalizations. The authors can also live image a fluorescently (f)-tagged VE-cadherin (maybe with another f-tagged rab5) and assess their association dynamics in IFT20 KD cells (similar to fig6C).

    Thank you for this helpful suggestion.

    Planned 4: Immunofluorescence staining for EEA1 (early endosome), RAB7 (late endosome), RAB4 (fast recycling), RAB11 (recycling endosome) along with VE-cadherin in IFT20 WT and KD cells to determine its localization pattern.

    Planned 5: Additional colocalization analysis such as adding intensity profiles and possibly proximity ligation assay.

    Beyond the scope of this manuscript 1: While we agree that imaging the dynamics of FP-tagged VE-cadherin in live cells would provide more detail about its localization, we feel that this is beyond the scope of the current manuscript.

    These experiments will determine the localization of VE-cadherin across various endosomal compartments and strengthen the current colocalization data.

    • Primary cilia do seem to regulate vascular plexus in the mouse retina as well as endothelial permeability through mediating subcellular localization of junction proteins. The authors do not clearly exclude the ciliary function of IFT20 in mediating lymphatic endothelial cell-cell junctions. A rescue experiment can help settle this question by targeting IFT20 exclusively to cilia (or not) and assessing, for example, VE-cadherin localization. The following is optional: It is also unclear whether the described regulation is specific to IFT20 or can be phenocopied by the ablation of another IFT subunit and/or cilia ablation through the depletion of a non-IFT cilia assembly regulator.

    Thank you for this helpful suggestion. We propose an alternative strategy.

    Planned 6: To determine the role of ciliary vs. nonciliary functions, we will knockdown IFT74, an IFT protein in the same IFT complex B as IFT20 that is required for cilia assembly and function but is not known to participate in vesicular trafficking. We will assess VE-cadherin localization in IFT74 WT and KD cells by immunofluorescence.

    Beyond the scope of the manuscript 2: We have not optimized reagents for targeting IFT20 to the cilium (e.g. ciliary targeting sequence) and believe that assessing the effects of a protein from the same IFT complex (IFT74) without known nonciliary functions will alleviate the reviewer’s concern.

    • Figs. 7A and B do not seem very convincing. The control vs. IFT20 KD western blot levels look mostly similar between the two conditions. The result section does not translate the actual data in Fig. 7A and B. Additionally, there are no statistical comparisons between control and KD conditions in the graphs. Except for a potential pVEGFR-3 increase at 30 min VEGF-C in IFT20 KD cells, but after washout the level is similar to control. This figure does not support well the model presented in fig. 8. The conclusion in lines 456-459 has to be toned down.

    Thank you for this helpful suggestion.

    Planned 7: We will remove these western blot data with the exception of pVEGFR-3 and add phospho-tyrosine immunofluorescence. We will use immunofluorescence to quantify phosphorylated tyrosine levels and repeat western blots for pVEGFR-3 at different concentrations and time points of VEGF-C stimulation in IFT20 WT and KD cells. We will remove the other western blot data and revise the text accordingly. We will also attempt to pull down total VEGFR-3 and then blot for pVEGFR-3 to improve sensitivity of this assay.

    These experiments will focus our analysis on the activation of VEGFR-3.

    • The authors were not able to stain for pVEGFR3. It would still be helpful to see a colocalization between total VEGFR3, IFT20, and VE-cadherin in control cells and IFT20 KD cells (VEGFR3 and VE-cadherin).

    Thank you for this helpful suggestion.

    Planned 8: We will perform immunofluorescence for VEGFR-3, IFT20, and VECAD and assess their localization.

    Minor comments

    • The control used in Figures 1 and 2 does not seem ideal. The proper control would be IFT20fl/fl cre neg. Is there a reason why the authors excluded a lox allele in control? Also, the authors have to provide the mice age used in these figures and when the Cre kicks in in the result section.

    Thank you for this helpful suggestion.

    Planned 9: We will clarify the use of control genotypes, and add mouse ages and Cre details to results/methods. This is a constitutive LYVE-1 Cre.

    • Please describe the overall mouse phenotype(s) of the LYVE1 CRE-IFT20 flox.

    Thank you for pointing out this oversight.

    Planned 10: We will include a description of the overall phenotypes of LYVE1 Cre IFT20 KOs in the text. One notable phenotype is abdominal ascites.

    • Line 109:'By expression, the authors probably mean immunostained.

    Thank you for pointing out this oversight.

    Planned 11: We will change to “immunostaining for”.

    • Many graphs exhibit undefined Y-axis labels and units. Please clarify these as well as the way they were quantified. Include such information in figure legends and/or in the materials and methods section. The figures in question are fig1C, E and F, fig2E, fig3E, fig4b and D, fig6b and D, fig7B and C.

    Thank you for this helpful suggestion.

    Planned 12: We will clarify the quantification strategies and units in the text and figure legends and make sure the axes are clearly labeled.

    • Line 295:'homeostasis"-the authors probably mean in a serum-rich condition.

    Thank you for this helpful suggestion.

    Planned 13: That is indeed what we meant. We will merge this sentence and the next sentence to be clearer.

    • Fig4C specifically the lower two images on the right side: the images do not seem to represent the corresponding graphs.

    Thank you for this helpful suggestion.

    Planned 14: We will double check these images and adjust if necessary.

    • Please add the statistical tests used to evaluate significance in all figure legends.

    Thank you for pointing out this oversight.

    Planned 15: We will be sure statistical tests are named in all figure legends.

    Reviewer #1 (Significance (Required)):

    This study provides novel insights into IFT20's role in VE-cadherin trafficking and endothelial junction stability, with its strongest aspect being the in vivo data in Figures 1 and 2, demonstrating lymphatic defects upon IFT20 loss. This represents a conceptual advance by extending IFT protein function beyond cilia (if one of the major comments is addressed) to vascular integrity. However, mechanistic depth is lacking, and ciliary role was not tested-additional rescue and colocalization experiments are needed to confirm the model. The study will interest vascular and lymphatic biologists, as well as cell biologists studying intracellular trafficking and cilia.

    Expertise: cilia and mouse genetics

    __Reviewer #2 __

    (Evidence, reproducibility and clarity (Required)):

    Paulson et al. use an in vivo model of IFT20 deletion (Lyve1-Cre) and primary lymphatic endothelial cell (LEC) cultures to investigate the role of IFT20 in controlling LEC-LEC junction dynamics. The key findings/suggestions include: i) Authors show alterations in the VE-cadherin (or ZO-1) staining at the LEC junctions upon IFT20 deletion or silencing. ii) They also show evidence of the IFT20 localization to RAB5 endosomes and alteration of RAB5 endosome dynamics upon IFT20 silencing.

    In the current manuscript, some of the key data are not convincing. Further experimentation and analysis (also of the existing data) are needed to solidify the authors' statements as detailed below. I expect that the suggested experiments can be executed in 3-to-6 months and require, at least, antibodies, which have not been used in the current manuscript.

    Major comments

    1. The data information, presented in the figure legends, is difficult to understand. The authors should always indicate how many biological replicates and independent experiments the data is derived from. This holds also for the representative images. Now, it seems that some of the quantified data are derived from only 1 experiment (see, for example, rows 423-425: "Graphs show one representative biological replicate of two, each comprising two technical replicates with 100+ cells per condition"). The quantifications should be based on data from at least three independent experiments.

    Often data points represent the field of views from a single sample, thus, biasing the statistical testing. The data points should represent biological replicates or independent experiments to allow the reader to make conclusions, about whether the findings are statistically significant and can be repeated.

    Thank you for this helpful critique.

    Planned 16: We will be sure to indicate biological and technical replicates and ensure that quantifications are representative of at least three independent experiments. We will also ensure that quantifications are statistically robust.

    The Lyve1-Cre is not specific for lymphatic vasculature (for example https://www.jax.org/strain/012601# and Lee LK et al. 2020, Cell Reports), as also stated by the authors (row 112). However, this is not shown in the data and complicates the interpretation of the data. Here, authors can stain the IFT20 with their existing mouse IFT20-specific antibody to show the loss in the lymphatic and/or blood vasculature. If IFT20 is lost in both vasculature types, it is not possible to say "lymphatic specific" (for example, row 143) and draw conclusions that the observed phenotypes would be primary to IFT20 loss in the lymphatic vasculature.

    Thank you for this helpful suggestion.

    Planned 17: We will assess IFT20 KO in blood vasculature and tone down lymphatic-specific language in the text.

    The authors write (rows 164-168) "Lymphatic vessels in the IFT20 KO or VE-cadherin KO embryonic dorsal skin exhibited increased and variable lumen size and excessive branching, suggesting that impaired lymphatic organization and function contributed to the fluid homeostasis defect. Here, immunofluorescence staining for LYVE-1 in the ear skin revealed similar patterning defects in adult IFT20 KO lymphatic vessels (Figure 2A), that have also been described in VE-cadherin KO mice (Hägerling et al., 2018)." However, based on Figure 2A, it is not obvious that there would be excessive lymphatic vessel branching, impaired organization or similarities to VE-cadherin deleted lymphatic vessels. To justify their statement, the authors should provide quantification of the branching (at least 3 mice/genotype).

    Thank you for this helpful critique.

    Planned 18: Based on the suggestion from Reviewer 3, we will remove these morphological and skin drainage data.

    IFT20 deletion or silencing causes alterations in the cell junction pattern/VE-cadherin intensity. The authors' interpretation that IFT20 deletion/silencing would cause discontinuous or "button-like" junctions is not supported by the provided images (Figures 1E, 3F, 6A, 6C). Rather, it seems that the levels of VE-cadherin in vivo are decreased, whereas the "continuity" of the junction is not altered. In cell culture, IFT20 silencing seems to cause wider and, to some extent, overlapping VE-cadherin junctions and not "discontinuous". These junctions may represent a more immature state. The authors should change the nomenclature accordingly or provide additional data. Using the existing cell culture experiment images, it would be more appropriate to analyze the width of the VE-cadherin junctions, instead of the "granularity".

    Thank you for this helpful suggestion.

    To assess VE-cadherin levels in vitro, we will perform western blots as described in Planned 1 above.

    Planned 19: We will measure widths of junctions from IFT20 KD and WT images and adjust the language in the text.

    Paulson et al. show images of IFT20 and RAB5 double-stained samples. The co-localization seems to happen mostly at the weakly IFT20 positive puncta (Figure 3A-B). Authors should show the disappearance of the signal in the siIFT20 treated samples (in comparison to siControl samples) to highlight the specificity of the weak signal.

    Thank you for this helpful suggestion.

    Planned 20: We will add data showing the IFT20 KD more clearly at high magnification.

    1. The Authors analyze the co-localization of VE-cadherin and RAB5 as co-localization area (Figure 6C-D). The images show that the co-localization is stated to happen at LEC periphery/junctions. LEC periphery is notoriously thin and microscope Z-resolution does not allow distinction of truly co-localizing or "on top of each other" signal. Based on row 607 co-localization would be expected to happen at least in EEA1+ vesicles, which are located perinuclearly (not at the junctions) in LECs (Korhonen et al. 2022, JCI). Authors could use EEA1, RAB5, and VE-cadherin triple staining for the quantification.

    Thank you for this helpful suggestion.

    Please see Planned 3 and Planned 4 above where we propose experiments to address this concern.

    In the current experiments, authors cannot conclude whether the VE-cadherin signal is at the cell junction (non-internalized), in endosomes (internalized during the experiment), or newly produced VE-cadherin on its way to the plasma membrane. To allow conclusions about the internalized VE-cadherin, and its localization in RAB5 vesicles, authors should conduct, for example, a classical endocytosis assay: incubation of live cells with non-blocking anti-VE-cadherin antibody, followed by acid wash to remove the non-internalized antibody, fixation and staining for RAB5. Also, shorter VEGF-C treatment would allow conclusions about the VE-cadherin dynamics.

    Thank you for this helpful suggestion.

    In Planned 2 above, we will perform immunofluorescence staining for cell-surface VE-cadherin using permeabilized and non-permeabilized IFT20 WT and KD cells during VEGF-C stimulation at various timepoints and washout to address this concern.

    siRNAs can have off-target effects and, thus, the use of at least two independent methods/oligos for silencing is needed. Paulson et al. use a pool of 4 oligos for silencing. They should rather test the efficacy of the single oligos and then use the two best oligos (1/sample) to show and quantify the same phenotype. This is needed at least for the key experiments shown in Figures 4C-D, Figure 6A-B (see also comment #3), Figure 7A-B

    Thank you for this helpful suggestion. We chose these reagents based on pooled siRNAs at low concentration minimizing off-target effects while still achieving strong KD vs. single siRNAs at higher concentration. Please see this technical note for further information about minimizing off-target effects by the use of pooled siRNAs vs. single siRNAs: https://horizondiscovery.com/-/media/Files/Horizon/resources/Application-notes/off-target-tech-review-technote.pdf?sc_lang=en

    1. “SMARTpool siRNA reagents pool four highly functional SMARTselection designed siRNAs targeting the same gene. Studies show that strong on-target gene knockdown can be achieved with minimal off-target effects if a pool consisting of highly functional multiple siRNA is subsituted for individual duplexes. This finding is in contrast to speculation that mixtrues of siRNAs can compound off-target effects. … [Their data show that] while individual duplexes delivered at 100 nM can induce varying numbers of off-targeted genes, transfection of the corresponding SMARTpool siRNA (100 nM total concentration) induces only a fraction of the total off-target profile.”
    2. “Our scientists have identified a unique combination of [chemical] modifications that eliminate as much as 80% of off-target effects.”
    3. “The ON-TARGET*plus *product line is comprised of four individual siRNAs, and SMARTpool reagents which are chemically modified and rationally designed to minimize off-target effects.”

    OPTIONAL: Paulson et al stated in the first article (2021, Front. Cell Dev. Biol.) that IFT20 deletion/silencing causes lymphatic endothelial phenotypes due to its role in primary cilia, whereas here the authors conclude that IFT20 controls VE-cadherin dynamics at the RAB5 vesicles. However, the current experiments cannot dissect the role of IFT20 in these two distinct locations. For this, authors could delete/silence another gene required for primary cilia or RAB5 endosomes and then analyze, which IFT20 phenotypes are recapitulated.

    Thank you for this helpful suggestion. Please see Planned 6 above where we propose to determine the role of ciliary vs. nonciliary IFT functions by knocking down IFT74, an IFT protein in the same IFT complex B as IFT20 that is required for cilia assembly and function but is not known to participate in vesicular trafficking. We will assess VE-cadherin localization in IFT74 WT and KD cells by immunofluorescence.

    The data shown in Figure 2 B-E (Lymphatic drainage) is not necessary for the current manuscript ("IFT20 regulates VE-cadherin traffic in LECs") and can be removed. As the authors state in the manuscript, the drainage phenotype may be due to lymphatic vessel valve defects (rows 584-585) rather than primary for LEC-LEC junction defects. The data does not justify the abstract sentence "and lymph transport is impaired by intracellular sequestration of VE-cadherin" (row 42).

    Thank you for this helpful suggestion. Please see Planned 18 above, where we propose to remove these data.

    Minor comments

    1. For some of the images, the signal should be enhanced to allow visual inspection also in the paper version (Figures 5A-B and 6C, magenta).

    Thank you for this helpful suggestion.

    Planned 21: We will enhance the signal in the indicated figures.

    Authors show representative Western Blots and quantification of several biological replicates/sample types to investigate signaling responses upon VEGF-C treatment of control and siIFT20 cells. The authors state that the P-levels of VEGFR3, ERK, VE-cadherin, and AKT have different dynamics in control and IFT20-silenced cells. To justify this conclusion, authors should test the statistical significance between the siControl and siIFT20 samples at each time point. The current quantification (Figure 7B) shows that there is, at least, a trend of increased p-VEGFR3, p-VE-cadherin, p-ERK, and p-AKT in IFT20 silenced cells. However, the representative Western Blot image does not display a clear difference (Figure 7A). Authors should include the original western blots, used for quantification, as supplements.

    Thank you for this helpful suggestion. Please see Planned 7 above where we propose to remove these data with the exception of pVEGFR-3 and add corresponding immunofluorescence data. We will ensure blots are included as supplemental figures.

    The authors use western blot quantification to show that the altered LEC junctions affect VEGFR3 signaling. They further hypothesize that the increased VEGFR3 signaling may be a consequence of VEGFR3 localization in endosomes. The authors did not detect any signal using the phospho-specific VEGFR3 antibody (rows 441-442). To analyze the location of VEGFR3 upon VEGF-C treatment in siControl and siIFT20 LECs, the authors should use anti-VEGFR3 (total) antibodies that have been shown to detect VEGFR3 in similar assays.

    Thank you for this helpful suggestion.

    Please see Planned 8 above where we will perform immunofluorescence for VEGFR-3, IFT20, and VECAD and assess their localization.

    The normality of the data should be tested before the selection of the statistical test. If this has been done, please, indicate it in the materials and methods or re-run the statistical analysis, if some of the data is not normally distributed.

    Thank you for this helpful suggestion.

    Planned 22: We will double check the statistics and normality for all quantifications.

    The authors should use arrows, arrowheads, etc. to highlight examples of relevant features in the images. For example, in Figure 3C, the increased stress fiber formation is not obvious to the reader.

    Thank you for this helpful suggestion.

    Planned 23: We will add arrows etc. where appropriate.

    Reviewer #2 (Significance (Required)):

    Lymphatics are essential for fluid, leukocyte, and lipid trafficking to lymph nodes and/or systemic circulation. Recent findings have promoted lymphatics as a potential target to control the level of adaptive immunity in inflammation-associated diseases, including tumorigenesis (for example Song et al 2020, Nature). Early work on lymphatic endothelium in vivo, highlighted the dynamics of lymphatic endothelial junction, which, reversibly, can alter between continuous and discontinuous ("button-like") states (Baluk 2007, Am. Jour. Pathol.; Yao 2012, Am J. Pathol.). These changes may have an effect on fluid drainage capacity, lymphatic vessel growth, and prevention of pathogen dissemination to the systemic circulation. Recently, lymphatic junctions have been shown to present hubs of VEGFR3 signaling, VEGFR3 and VE-cadherin dynamics, and leukocyte transmigration (Sung et al. 2022, Nat. Cardiovasc. Res.; Hagerling et al. 2018, EMBO J.; Liaqat et al. 2024, EMBO J.). Thus, the manuscript by Paulson et al. investigates a topical subject.

    The authors suggest a role for IFT20 in the control of VE-cadherin dynamics. Based on my expertise in lymphatic endothelial biology, I envision that the manuscript can potentially increase knowledge on the regulators of the lymphatic endothelial junctions, which might have physiological, and in the long term, translational significance. However, in the current manuscript, the exact mechanisms of how IFT20 controls lymphatic endothelial junctions are left open. In addition to the lymphatic research field, the study is, potentially of interest to researchers working on blood vasculature or, even, epithelium, i.e. tissues where junctional dynamics play a major role in health and disease.

    Furter controls, analysis, and experimentation are needed to warrant the authors' statements. In their future work, the authors should also consider means to rigorously dissect the IFT20 functions in primary cilia and endosomes.

    __Reviewer #3 __

    (Evidence, reproducibility and clarity (Required)):

    In this manuscript, the group of Fink and coworkers investigates mechanistic aspects of the intraflagellar transport protein 20 (IFT20) function in lymphatic endothelial cells (LECs). In a previous study, this group had demonstrated the presence of primary cilia on LECs and shown that loss of IFT20 during development resulted in edema, lymphatic vessel dilation and altered branching. Lymphatic-specific deletion of IFT20 cell-autonomously exacerbated acute lymphangiogenesis after corneal suture. In this manuscript, Paulson et al. recapitulate the suture-induced hyper-lymphangiogenesis after lymphatic-specific IFT20 KO using a LYVE1-Cre delete strain and demonstrate a reduced, more discontinuous VE-Cadherin (VECad) staining in newly formed lymphatic vessels (LVs). Prompted by distended and hyperbranching dermal vessels, the performed functional tracer injection experiments and demonstrate increased lymphatic backflow and leakage into the interstitium. To gain further mechanistic insights the authors turned to reductionist cell culture models, starting with a mouse LEC line, in which IFT20 had been deleted using CRISPR/Cas9 resulting in loss of primary cilia, increased stress fibre formation and impaired junctional integrity. More importantly, similar effects were detected in human dermal (HD)LECs after IFT20 KD. Further IFT20 KD HDLECs showed accumulation of RAB5+ vesicles indicating defective endosome maturation. Indistinguishable formation of RAB5+ endosomes after VEGF-C stimulation in HDLECs and IFT20KD HDLECs indicated that endocytosis and formation of early endosomes occur independent if IFT20. Through starvation, stimulation and wash-out experiments the authors provide colocalization data suggesting that after VEGF-C stimulation IFT20 is recruited to endosomes where it contributes to VECad recycling. Finally, the authors addressed if the increase in RAB5+ endosomes following VEGF-C stimulation resulted in prolonged retention of signaling-active VEGFR-3 in endosomes. Western blotting for phosphorylation of VEGFR-3 and its downstream signaling components after activation of starved HDLECs or IFT20KD HDLECs and subsequent factor wash out provided evidence towards this model.

    Subsequently open question and potential suggestions for improvement are listed: The authors describe a slight leakiness of the LYVE1-Cre deleter strain to result in massive hemangiogenesis (line112). How extensive is the resulting deletion in blood endothelial cells? What are the consequences for VECad distribution in BEC junctions i.e. for blood vessels and vascular permeability? Are the defects described specific for LECs or are the manifestation of generic defects in LECs?

    Thank you for these helpful suggestions.

    Please see Planned 17 above where we will assess IFT20 KO in blood vasculature and tone down lymphatic-specific language in the text.

    Fig. 1 E, what is the distribution of LYVE1 in IFT20 KO LECs at higher magnification, is LYVE1 excluded from the VECad expression domain?

    Thank you for this helpful suggestion.

    Planned 24: We will review our corneal confocal data to address this question.

    Fig.1 F, what does VECad-positive LV (%) area (line 154 - 155) refer to, given that all LECs are VECad+ but the junctional distribution of the protein is distinctly different?

    Thank you for pointing out our need to clarify. This quantification measures the overlap of VE-cadherin with LYVE-1 as a way to measure the area covered by adherens junctions between lymphatic endothelial cells. Where junctions are punctate, they have smaller area vs. long continuous junctions.

    Planned 25: We will update the text to clarify this measurement.

    In the discussion, the authors speculate that the development of valves could be potentially impaired in IFT20 LEC KO mice. Ear skin would be an excellent tissue to stain the valves and analyse their structure in collecting LVs. Of particular interest in this context are Int a9, VECad, FOXC2 and PROX1 expression. The later two are required for valve formation and upregulated in valve forming areas in response to oscillatory shear stress (Sabine A et al. (2012) Dev Cell 22 (2):430-445. doi:10.1016/j.devcel.2011.12.020).

    Thank you for this helpful suggestion. Based on the suggestion from Reviewer 2, we will remove the ear lymph drainage data and focus on the cell biology in this manuscript. Our current experiments focus more on lymphatic valve formation in this context and these data can be moved to a separate manuscript.

    Planned 26: We will revise the text to remove speculation about valve development in this model and address this in a later manuscript.

    Does IFT20 KO and loss of the primary cilium impair OSS sensing and result in a failure to express sufficient levels of PROX1 for valve formation (Fig. 7 C).

    Thank you for this helpful comment. We will address the role of cilia in OSS sensing and valve formation in a forthcoming manuscript.

    A larger area view including pre-collectors and collectors would be informative and reveal changes in the overall structure of the lymphatic vessel bed in absence of IFT20.

    Based on the suggestion from Reviewer 2, we will remove these data.

    Fig. 2 A, (line 187 - 190) please indicate the age of analysed animals.

    Planned 27: We will add the ages of mice used.

    With respect to Fig.1, LVs in the area are mainly capillaries, what is the distribution of VECad? Are the LVs comprised of oak-leave shaped LECs, higher magn. pictures would be required.

    Thank you for this helpful suggestion.

    Planned 28: We will include higher magnification images of capillaries.

    Fig. 2 (C - E) Line 201 - 203 the description of retrograde flow using a clock terminology is unusual and not clear to the reader. Is this meant relative to the point of injection with 12 being at the top or relative to the injection axis (i.e. forward / backward direction)? It would seem that indication of the angle in combination with a sketch of the analysis would help the reader to interpret these data.

    Thank you for this helpful critique. We will remove these data based on this suggestion and that of Reviewer 2.

    The application of cell culture models is appropriate, however, the value of the mLEC model is questionable given that VECad is not detectable in these cells and PROX1 and VEGFR-3 staining are not shown. Therefore, the HDLEC model bears significantly more relevance. In Fig. 3D, were mLECS mitotically arrested during the 24hrs transwell migration, to exclude division and crowding effects during the observation time?

    Thank you for this helpful critique.

    Planned 29: We will clarify the methods for this experiment in the text.

    Fig. 6 It is commendable that the authors report their lack of success to directly visualize VEGFR-3 endocytosis by IF and attempt a WB analysis instead. However given the spread of the results normalization to ß-actin as a loading control appears inappropriate. Phosphorylated forms of VEGFR-3 and VECad should be normalized to the expression of the total protein as measured with a non-phospospecific antibody, exactly the way done here for ERK1/2 and AKT. Generally, IP-WB experiments provide superior data in this type of setting.

    Thank you for this helpful suggestion. Based on suggestions from the other reviewers, we will remove these WB data with the exception of pVEGFR-3 and add corresponding immunofluorescence. We will include additional time points and include blots used for quantifications as supplements.

    Line 597 - 599: "VEGFR-3 signaling is required for the establishment of VE-cadherin button junctions as lymphatic collecting vessels mature but is not required for their maintenance (Jannaway et al., 2023)." Collecting LVs are characterized by zipper junctions, but not button junctions. Therefore, this sentence needs clarification.

    Thank you for this helpful suggestion.

    Planned 30: We will clarify this text.

    Reviewer #3 (Significance (Required)):

    The role of IFT20 in formation of the primary cilium and endocytic vesicle transport warrants its investigation in lymphatic endothelial cells. Therefore, this study addresses relevant questions and provides important first insights into the cell biological function of IFT20 in this cell type. IFT20 has so far not been implicated in endocytosis and recycling of VECad and VEGFR-3 and the model suggested by the authors is compelling and adds to the mechanistic understanding of previous studies on the role of VECad in LECs. In particular, it could be of relevance for the enigmatic formation of button junction in lymphatic capillaries and the mechano-response of LECS underlying valve formation. At this point, the picture obtained from the endocytosis assays is more conclusive compared to the analysis of the impact of IFT20 loss on button junction formation. Clearly the study is of interest for a general cell biological audience as well as vascular biologists.

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    Referee #3

    Evidence, reproducibility and clarity

    In this manuscript, the group of Fink and coworkers investigates mechanistic aspects of the intraflagellar transport protein 20 (IFT20) function in lymphatic endothelial cells (LECs). In a previous study, this group had demonstrated the presence of primary cilia on LECs and shown that loss of IFT20 during development resulted in edema, lymphatic vessel dilation and altered branching. Lymphatic-specific deletion of IFT20 cell-autonomously exacerbated acute lymphangiogenesis after corneal suture. In this manuscript, Paulson et al. recapitulate the suture-induced hyper-lymphangiogenesis after lymphatic-specific IFT20 KO using a LYVE1-Cre delete strain and demonstrate a reduced, more discontinuous VE-Cadherin (VECad) staining in newly formed lymphatic vessels (LVs). Prompted by distended and hyperbranching dermal vessels, the performed functional tracer injection experiments and demonstrate increased lymphatic backflow and leakage into the interstitium. To gain further mechanistic insights the authors turned to reductionist cell culture models, starting with a mouse LEC line, in which IFT20 had been deleted using CRISPR/Cas9 resulting in loss of primary cilia, increased stress fibre formation and impaired junctional integrity. More importantly, similar effects were detected in human dermal (HD)LECs after IFT20 KD. Further IFT20 KD HDLECs showed accumulation of RAB5+ vesicles indicating defective endosome maturation. Indistinguishable formation of RAB5+ endosomes after VEGF-C stimulation in HDLECs and IFT20KD HDLECs indicated that endocytosis and formation of early endosomes occur independent if IFT20. Through starvation, stimulation and wash-out experiments the authors provide colocalization data suggesting that after VEGF-C stimulation IFT20 is recruited to endosomes where it contributes to VECad recycling. Finally, the authors addressed if the increase in RAB5+ endosomes following VEGF-C stimulation resulted in prolonged retention of signaling-active VEGFR-3 in endosomes. Western blotting for phosphorylation of VEGFR-3 and its downstream signaling components after activation of starved HDLECs or IFT20KD HDLECs and subsequent factor wash out provided evidence towards this model.

    Subsequently open question and potential suggestions for improvement are listed: The authors describe a slight leakiness of the LYVE1-Cre deleter strain to result in massive hemangiogenesis (line112). How extensive is the resulting deletion in blood endothelial cells? What are the consequences for VECad distribution in BEC junctions i.e. for blood vessels and vascular permeability? Are the defects described specific for LECs or are the manifestation of generic defects in LECs? Fig. 1 E, what is the distribution of LYVE1 in IFT20 KO LECs at higher magnification, is LYVE1 excluded from the VECad expression domain? Fig.1 F, what does VECad-positive LV (%) area (line 154 - 155) refer to, given that all LECs are VECad+ but the junctional distribution of the protein is distinctly different?

    In the discussion, the authors speculate that the development of valves could be potentially impaired in IFT20 LEC KO mice. Ear skin would be an excellent tissue to stain the valves and analyse their structure in collecting LVs. Of particular interest in this context are Int a9, VECad, FOXC2 and PROX1 expression. The later two are required for valve formation and upregulated in valve forming areas in response to oscillatory shear stress (Sabine A et al. (2012) Dev Cell 22 (2):430-445. doi:10.1016/j.devcel.2011.12.020). Does IFT20 KO and loss of the primary cilium impair OSS sensing and result in a failure to express sufficient levels of PROX1 for valve formation (Fig. 7 C). A larger area view including pre-collectors and collectors would be informative and reveal changes in the overall structure of the lymphatic vessel bed in absence of IFT20. Fig. 2 A, (line 187 - 190) please indicate the age of analysed animals. With respect to Fig.1, LVs in the area are mainly capillaries, what is the distribution of VECad? Are the LVs comprised of oak-leave shaped LECs, higher magn. pictures would be required. Fig. 2 (C - E) Line 201 - 203 the description of retrograde flow using a clock terminology is unusual and not clear to the reader. Is this meant relative to the point of injection with 12 being at the top or relative to the injection axis (i.e. forward / backward direction)? It would seem that indication of the angle in combination with a sketch of the analysis would help the reader to interpret these data.

    The application of cell culture models is appropriate, however, the value of the mLEC model is questionable given that VECad is not detectable in these cells and PROX1 and VEGFR-3 staining are not shown. Therefore, the HDLEC model bears significantly more relevance. In Fig. 3D, were mLECS mitotically arrested during the 24hrs transwell migration, to exclude division and crowding effects during the observation time?

    Fig. 6 It is commendable that the authors report their lack of success to directly visualize VEGFR-3 endocytosis by IF and attempt a WB analysis instead. However given the spread of the results normalization to ß-actin as a loading control appears inappropriate. Phosphorylated forms of VEGFR-3 and VECad should be normalized to the expression of the total protein as measured with a non-phospospecific antibody, exactly the way done here for ERK1/2 and AKT. Generally, IP-WB experiments provide superior data in this type of setting. Line 597 - 599: "VEGFR-3 signaling is required for the establishment of VE-cadherin button junctions as lymphatic collecting vessels mature but is not required for their maintenance (Jannaway et al., 2023)." Collecting LVs are characterized by zipper junctions, but not button junctions. Therefore, this sentence needs clarification.

    Significance

    The role of IFT20 in formation of the primary cilium and endocytic vesicle transport warrants its investigation in lymphatic endothelial cells. Therefore, this study addresses relevant questions and provides important first insights into the cell biological function of IFT20 in this cell type. IFT20 has so far not been implicated in endocytosis and recycling of VECad and VEGFR-3 and the model suggested by the authors is compelling and adds to the mechanistic understanding of previous studies on the role of VECad in LECs. In particular, it could be of relevance for the enigmatic formation of button junction in lymphatic capillaries and the mechano-response of LECS underlying valve formation. At this point, the picture obtained from the endocytosis assays is more conclusive compared to the analysis of the impact of IFT20 loss on button junction formation. Clearly the study is of interest for a general cell biological audience as well as vascular biologists.

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    Referee #2

    Evidence, reproducibility and clarity

    Paulson et al. use an in vivo model of IFT20 deletion (Lyve1-Cre) and primary lymphatic endothelial cell (LEC) cultures to investigate the role of IFT20 in controlling LEC-LEC junction dynamics. The key findings/suggestions include: i) Authors show alterations in the VE-cadherin (or ZO-1) staining at the LEC junctions upon IFT20 deletion or silencing. ii) They also show evidence of the IFT20 localization to RAB5 endosomes and alteration of RAB5 endosome dynamics upon IFT20 silencing.

    In the current manuscript, some of the key data are not convincing. Further experimentation and analysis (also of the existing data) are needed to solidify the authors' statements as detailed below. I expect that the suggested experiments can be executed in 3-to-6 months and require, at least, antibodies, which have not been used in the current manuscript.

    Major comments

    1. The data information, presented in the figure legends, is difficult to understand. The authors should always indicate how many biological replicates and independent experiments the data is derived from. This holds also for the representative images. Now, it seems that some of the quantified data are derived from only 1 experiment (see, for example, rows 423-425: "Graphs show one representative biological replicate of two, each comprising two technical replicates with 100+ cells per condition"). The quantifications should be based on data from at least three independent experiments.

    Often data points represent the field of views from a single sample, thus, biasing the statistical testing. The data points should represent biological replicates or independent experiments to allow the reader to make conclusions, about whether the findings are statistically significant and can be repeated.

    1. The Lyve1-Cre is not specific for lymphatic vasculature (for example https://www.jax.org/strain/012601# and Lee LK et al. 2020, Cell Reports), as also stated by the authors (row 112). However, this is not shown in the data and complicates the interpretation of the data. Here, authors can stain the IFT20 with their existing mouse IFT20-specific antibody to show the loss in the lymphatic and/or blood vasculature. If IFT20 is lost in both vasculature types, it is not possible to say "lymphatic specific" (for example, row 143) and draw conclusions that the observed phenotypes would be primary to IFT20 loss in the lymphatic vasculature.
    2. The authors write (rows 164-168) "Lymphatic vessels in the IFT20 KO or VE-cadherin KO embryonic dorsal skin exhibited increased and variable lumen size and excessive branching, suggesting that impaired lymphatic organization and function contributed to the fluid homeostasis defect. Here, immunofluorescence staining for LYVE-1 in the ear skin revealed similar patterning defects in adult IFT20 KO lymphatic vessels (Figure 2A), that have also been described in VE-cadherin KO mice (Hägerling et al., 2018)." However, based on Figure 2A, it is not obvious that there would be excessive lymphatic vessel branching, impaired organization or similarities to VE-cadherin deleted lymphatic vessels. To justify their statement, the authors should provide quantification of the branching (at least 3 mice/genotype).
    3. IFT20 deletion or silencing causes alterations in the cell junction pattern/VE-cadherin intensity. The authors' interpretation that IFT20 deletion/silencing would cause discontinuous or "button-like" junctions is not supported by the provided images (Figures 1E, 3F, 6A, 6C). Rather, it seems that the levels of VE-cadherin in vivo are decreased, whereas the "continuity" of the junction is not altered. In cell culture, IFT20 silencing seems to cause wider and, to some extent, overlapping VE-cadherin junctions and not "discontinuous". These junctions may represent a more immature state. The authors should change the nomenclature accordingly or provide additional data. Using the existing cell culture experiment images, it would be more appropriate to analyze the width of the VE-cadherin junctions, instead of the "granularity".
    4. Paulson et al. show images of IFT20 and RAB5 double-stained samples. The co-localization seems to happen mostly at the weakly IFT20 positive puncta (Figure 3A-B). Authors should show the disappearance of the signal in the siIFT20 treated samples (in comparison to siControl samples) to highlight the specificity of the weak signal.
    5. The Authors analyze the co-localization of VE-cadherin and RAB5 as co-localization area (Figure 6C-D). The images show that the co-localization is stated to happen at LEC periphery/junctions. LEC periphery is notoriously thin and microscope Z-resolution does not allow distinction of truly co-localizing or "on top of each other" signal. Based on row 607 co-localization would be expected to happen at least in EEA1+ vesicles, which are located perinuclearly (not at the junctions) in LECs (Korhonen et al. 2022, JCI). Authors could use EEA1, RAB5, and VE-cadherin triple staining for the quantification.

    In the current experiments, authors cannot conclude whether the VE-cadherin signal is at the cell junction (non-internalized), in endosomes (internalized during the experiment), or newly produced VE-cadherin on its way to the plasma membrane. To allow conclusions about the internalized VE-cadherin, and its localization in RAB5 vesicles, authors should conduct, for example, a classical endocytosis assay: incubation of live cells with non-blocking anti-VE-cadherin antibody, followed by acid wash to remove the non-internalized antibody, fixation and staining for RAB5. Also, shorter VEGF-C treatment would allow conclusions about the VE-cadherin dynamics.

    1. siRNAs can have off-target effects and, thus, the use of at least two independent methods/oligos for silencing is needed. Paulson et al. use a pool of 4 oligos for silencing. They should rather test the efficacy of the single oligos and then use the two best oligos (1/sample) to show and quantify the same phenotype. This is needed at least for the key experiments shown in Figures 4C-D, Figure 6A-B (see also comment #3), Figure 7A-B
    2. OPTIONAL: Paulson et al stated in the first article (2021, Front. Cell Dev. Biol.) that IFT20 deletion/silencing causes lymphatic endothelial phenotypes due to its role in primary cilia, whereas here the authors conclude that IFT20 controls VE-cadherin dynamics at the RAB5 vesicles. However, the current experiments cannot dissect the role of IFT20 in these two distinct locations. For this, authors could delete/silence another gene required for primary cilia or RAB5 endosomes and then analyze, which IFT20 phenotypes are recapitulated.
    3. The data shown in Figure 2 B-E (Lymphatic drainage) is not necessary for the current manuscript ("IFT20 regulates VE-cadherin traffic in LECs") and can be removed. As the authors state in the manuscript, the drainage phenotype may be due to lymphatic vessel valve defects (rows 584-585) rather than primary for LEC-LEC junction defects. The data does not justify the abstract sentence "and lymph transport is impaired by intracellular sequestration of VE-cadherin" (row 42).

    Minor comments

    1. For some of the images, the signal should be enhanced to allow visual inspection also in the paper version (Figures 5A-B and 6C, magenta).
    2. Authors show representative Western Blots and quantification of several biological replicates/sample types to investigate signaling responses upon VEGF-C treatment of control and siIFT20 cells. The authors state that the P-levels of VEGFR3, ERK, VE-cadherin, and AKT have different dynamics in control and IFT20-silenced cells. To justify this conclusion, authors should test the statistical significance between the siControl and siIFT20 samples at each time point.

    The current quantification (Figure 7B) shows that there is, at least, a trend of increased p-VEGFR3, p-VE-cadherin, p-ERK, and p-AKT in IFT20 silenced cells. However, the representative Western Blot image does not display a clear difference (Figure 7A). Authors should include the original western blots, used for quantification, as supplements.

    1. The authors use western blot quantification to show that the altered LEC junctions affect VEGFR3 signaling. They further hypothesize that the increased VEGFR3 signaling may be a consequence of VEGFR3 localization in endosomes. The authors did not detect any signal using the phospho-specific VEGFR3 antibody (rows 441-442). To analyze the location of VEGFR3 upon VEGF-C treatment in siControl and siIFT20 LECs, the authors should use anti-VEGFR3 (total) antibodies that have been shown to detect VEGFR3 in similar assays.
    2. The normality of the data should be tested before the selection of the statistical test. If this has been done, please, indicate it in the materials and methods or re-run the statistical analysis, if some of the data is not normally distributed.
    3. The authors should use arrows, arrowheads, etc. to highlight examples of relevant features in the images. For example, in Figure 3C, the increased stress fiber formation is not obvious to the reader.

    Significance

    Lymphatics are essential for fluid, leukocyte, and lipid trafficking to lymph nodes and/or systemic circulation. Recent findings have promoted lymphatics as a potential target to control the level of adaptive immunity in inflammation-associated diseases, including tumorigenesis (for example Song et al 2020, Nature). Early work on lymphatic endothelium in vivo, highlighted the dynamics of lymphatic endothelial junction, which, reversibly, can alter between continuous and discontinuous ("button-like") states (Baluk 2007, Am. Jour. Pathol.; Yao 2012, Am J. Pathol.). These changes may have an effect on fluid drainage capacity, lymphatic vessel growth, and prevention of pathogen dissemination to the systemic circulation. Recently, lymphatic junctions have been shown to present hubs of VEGFR3 signaling, VEGFR3 and VE-cadherin dynamics, and leukocyte transmigration (Sung et al. 2022, Nat. Cardiovasc. Res.; Hagerling et al. 2018, EMBO J.; Liaqat et al. 2024, EMBO J.). Thus, the manuscript by Paulson et al. investigates a topical subject.

    The authors suggest a role for IFT20 in the control of VE-cadherin dynamics. Based on my expertise in lymphatic endothelial biology, I envision that the manuscript can potentially increase knowledge on the regulators of the lymphatic endothelial junctions, which might have physiological, and in the long term, translational significance. However, in the current manuscript, the exact mechanisms of how IFT20 controls lymphatic endothelial junctions are left open. In addition to the lymphatic research field, the study is, potentially of interest to researchers working on blood vasculature or, even, epithelium, i.e. tissues where junctional dynamics play a major role in health and disease.

    Furter controls, analysis, and experimentation are needed to warrant the authors' statements. In their future work, the authors should also consider means to rigorously dissect the IFT20 functions in primary cilia and endosomes.

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    Referee #1

    Evidence, reproducibility and clarity

    The manuscript identified a novel role of Intraflagellar Transport Protein 20 (IFT20) in the function and homeostasis of lymphatic endothelial junctions. The authors showed that IFT20 regulates VE-cadherin localization at adherens junctions in lymphatic endothelial cells. The authors performed impressive in vivo work that shows the requirement for IFT20 for the homeostasis of intercellular junctions, lymphangiogenesis, and drainage function of lymphatic vessels. In contrast, the cell biology part of the paper was underwhelming and will need significant revisions to support the proposed model. In the result section, several conclusions have to be toned down to match the actual results. The study employs in vivo mouse models, immunofluorescence, biochemical assays, and loss-of-function experiments to support their conclusions.

    Major comments

    • The authors present disrupted localization of VE-cadherin. Is this a mislocalization and/or protein stability issue in IFT20 KD cells? A western blot can help assess protein levels, and a phase-chase endocytosis assay of VE-cadherin can strengthen evidence. The authors did not confirm the permeability phenotype seen in vivo.
    • While the authors focused on IFT20 and rab5, we do not have a clear idea about the vesicular dynamics as well as the status of early, late, and recycling endosomes in IFT20 KD cells. Is IFT 20 localized to non-rab5+ endosomes, and if yes, what are the species? A more general endosomal profiling would help strengthen the authors' message. For example, in Fig. 4-5, the authors will have to stain for other early endosomal markers as well as late, and recycling endosomal markers in control and IFT20 KD cells.
    • In fig. 6C, a majority of VE-cadherin is not associated with Rab5. Staining with additional endosomal markers might help identify other endosomal species colocalizing with VE-cadherin. It will be critical to add to Fig. 6c the intensity profiles depicting colocalizations. The authors can also live image a fluorescently (f)-tagged VE-cadherin (maybe with another f-tagged rab5) and assess their association dynamics in IFT20 KD cells (similar to fig6C).
    • Primary cilia do seem to regulate vascular plexus in the mouse retina as well as endothelial permeability through mediating subcellular localization of junction proteins. The authors do not clearly exclude the ciliary function of IFT20 in mediating lymphatic endothelial cell-cell junctions. A rescue experiment can help settle this question by targeting IFT20 exclusively to cilia (or not) and assessing, for example, VE-cadherin localization. The following is optional: It is also unclear whether the described regulation is specific to IFT20 or can be phenocopied by the ablation of another IFT subunit and/or cilia ablation through the depletion of a non-IFT cilia assembly regulator.
    • Figs. 7A and B do not seem very convincing. The control vs. IFT20 KD western blot levels look mostly similar between the two conditions. The result section does not translate the actual data in Fig. 7A and B. Additionally, there are no statistical comparisons between control and KD conditions in the graphs. Except for a potential pVEGFR-3 increase at 30 min VEGF-C in IFT20 KD cells, but after washout the level is similar to control. This figure does not support well the model presented in fig. 8. The conclusion in lines 456-459 has to be toned down.
    • The authors were not able to stain for pVEGFR3. It would still be helpful to see a colocalization between total VEGFR3, IFT20, and VE-cadherin in control cells and IFT20 KD cells (VEGFR3 and VE-cadherin).

    Minor comments

    • The control used in Figures 1 and 2 does not seem ideal. The proper control would be IFT20fl/fl cre neg. Is there a reason why the authors excluded a lox allele in control? Also, the authors have to provide the mice age used in these figures and when the Cre kicks in in the result section.
    • Please describe the overall mouse phenotype(s) of the LYVE1 CRE-IFT20 flox.
    • Line 109:'By expression, the authors probably mean immunostained.
    • Many graphs exhibit undefined Y-axis labels and units. Please clarify these as well as the way they were quantified. Include such information in figure legends and/or in the materials and methods section. The figures in question are fig1C, E and F, fig2E, fig3E, fig4b and D, fig6b and D, fig7B and C.
    • Line 295:'homeostasis"-the authors probably mean in a serum-rich condition.
    • Fig4C specifically the lower two images on the right side: the images do not seem to represent the corresponding graphs.
    • Please add the statistical tests used to evaluate significance in all figure legends.

    Significance

    This study provides novel insights into IFT20's role in VE-cadherin trafficking and endothelial junction stability, with its strongest aspect being the in vivo data in Figures 1 and 2, demonstrating lymphatic defects upon IFT20 loss. This represents a conceptual advance by extending IFT protein function beyond cilia (if one of the major comments is addressed) to vascular integrity. However, mechanistic depth is lacking, and ciliary role was not tested-additional rescue and colocalization experiments are needed to confirm the model. The study will interest vascular and lymphatic biologists, as well as cell biologists studying intracellular trafficking and cilia.

    Expertise: cilia and mouse genetics

  5. Version published to 10.1101/2025.01.15.631989v1 on bioRxiv