Multiple cis -regulatory elements control prox1a expression in distinct lymphatic vascular beds

Article activity feed

1. 

   Review Commons

   Oct 27, 2023

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

   Learn more at Review Commons

   ---

   Reply to the reviewers

   'The authors do not wish to provide a response at this time.'

   Read the original source
2. 

   Review Commons

   Oct 27, 2023

   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 presents the cis-regulatory analysis of the enhancers controlling prox1a gene in zebrafish. Authors used both evolutionary conservation and existing single-cell ATAC data to highlight the major role of two elements. I feel that the transgenesis work is quite solid and the main conclusions interesting. However, I feel the authors need to provide some extra validations for some of the analysis.

   1. the authors did not discuss the fact that euteleosts underwent an extra whole genome duplication and that prox1a might have a paralogue. They also perform genome alignment using non-duplicated outgroups (gar, xenopus) without …

   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 presents the cis-regulatory analysis of the enhancers controlling prox1a gene in zebrafish. Authors used both evolutionary conservation and existing single-cell ATAC data to highlight the major role of two elements. I feel that the transgenesis work is quite solid and the main conclusions interesting. However, I feel the authors need to provide some extra validations for some of the analysis.

   1. the authors did not discuss the fact that euteleosts underwent an extra whole genome duplication and that prox1a might have a paralogue. They also perform genome alignment using non-duplicated outgroups (gar, xenopus) without discussing. I am a bit skeptical about the use of mVISTA on relatively short expert of sequence aroudn a gene, as it is not able to capture the global molecular evolution parameters. I think the authors should also examine some of the precomputed phastCons / phylocons data performed and available on UCSC to confirm their findings. probably they should also examien a few more fish genome. I don't find this evolutioanry analysis extremely convinced and careful - which doesn't mean that the conclusions are wrong.
   2. I find the presentation, fairly obscure, the writing is quite convoluted, and the figures are very dense and not super explanatory, I would urge to improve (this is not helped by the fact that figure are their leged and presented at distinct places of this manuscript). For instance, I think having. a figure summarising signal from evolutionary conservation, scATAC and chromatin marks altogether would be quite essential.
   3. I also find the reanalysis of the single-cell ATAC described too scarcely: which are the genes used to identify the different cell populations?
   4. I feel the one additional experiment that the authors could have done would have been to use their construct to isolate the different cells population of interest and perform some regulatory profiling scuh as ATAC-seq or cut-and-tag on this population, to have a direct, in situ evidence of the activity of these regulatory elements.?

   I also feel that the evolutionary aspect could be discussed a bit more, what are the differences between the diffeerent vertebrate lineage, etc...

   (p7) active enhancer in a tissue: while ATAC gives a good indicated of accessibility it is not an indicate of activity as for instance H3K27Ac would be.

   Significance

   I think this is an interesting piece of work, which elaborates on previous studies on prox1a involvment in the lymphatic system but it doesn not bring essentially new perspective on the question.

   Read the original source
3. 

   Review Commons

   Oct 27, 2023

   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:

   Panara et al. identified the enhancer regions necessary for the tissue- and organ-specific expression of Prox1a, which is essential for lymphatic vessel development in zebrafish. The authors compared the sequences of eight species of osteichthyes, identified Conserved Non-Coding Elements (CNEs), and further predicted active enhancers by combining public databases. This sequence was analyzed using the ZED system. As a result, they confirmed that two out of ten sequences caused GFP expression in a subset of lymphatic endothelial cells. +15.2prox1a was required for the prox1a expression in the facial collecting lymphatics, …

   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:

   Panara et al. identified the enhancer regions necessary for the tissue- and organ-specific expression of Prox1a, which is essential for lymphatic vessel development in zebrafish. The authors compared the sequences of eight species of osteichthyes, identified Conserved Non-Coding Elements (CNEs), and further predicted active enhancers by combining public databases. This sequence was analyzed using the ZED system. As a result, they confirmed that two out of ten sequences caused GFP expression in a subset of lymphatic endothelial cells. +15.2prox1a was required for the prox1a expression in the facial collecting lymphatics, while -2.1prox1a was required in the facial lymphatic valves. They also predicted transcription factor binding to these enhancer regions and identified enhancer regions of -14, -71, -87Prox1a based on chromatin state. Additionally, they identified a core element within -2.1Prox1a, performed its loss-of-function experiments, and analyzed its phenotype, revealing the functional importance of these enhancer regions. The paper is logically well-structured and informative concerning the expression control of Prox1a.

   Major comments:

   • Are the key conclusions convincing?

   I think so.

   • Should the authors qualify some of their claims as preliminary or speculative, or remove them altogether?

   No need to qualify.

   • Would additional experiments be essential to support the claims of the paper? Request additional experiments only where necessary for the paper as it is, and do not ask authors to open new lines of experimentation.

   Figure 4H is probably incorrect. Looking at the morphology of the valve, it's inferred that the lymph flow goes from right to left. In this case, if the valve function is abnormal, the lymph flow that entered from the right should reflux back to the left. Is the reviewer misunderstanding something here? Clarity on this point could be provided with video images, similar to echocardiograms in mice.

   • Are the suggested experiments realistic in terms of time and resources? It would help if you could add an estimated cost and time investment for substantial experiments.

   Yes, they are realistic. I would like further clarification to ensure the data is correct.

   Minor comments:

   • Specific experimental issues that are easily addressable.

   It's important where Prox1 is expressed in the lymphatic system, but if the identified enhancers also regulate Prox1 expression in other tissues like the myocardium, it would be a significant finding. Do these enhancers have a role in controlling Prox1 expression in lymphatics and non-lymphatics?

   • Are prior studies referenced appropriately?

   Yes.

   • Are the text and figures clear and accurate?

   Yes.

   • Do you have suggestions that would help the authors improve the presentation of their data and conclusions?

   1. Regarding Figure 4G, the actual image is unknown due to the red shading. Can you discuss or clarify how knocking out the Prox1 enhancer affects valve formation? If Prox1 expression is reduced, does the normal extracellular matrix change? Why do these morphological changes occur?
   2. In mice, Prox1 is expressed in the heart valve endothelium. Have they checked the enhancer activity in other valves (heart valves or venous valves, if they exist)?
   3. -2.1prox1a is said to be important for Prox1 expression in the facial lymphatic valves. Are valves only formed in this part of the lymphatics in zebrafish at this stage? Why is it important to identify the enhancer for this particular valve? Are there no other lymphatic valves? Please explain in the text.
   4. They seem to emphasize +15.2's role in facial lymphatic expression. Compared to the trunk, why is it significant that there is a unique enhancer acting in this area? Is there something functionally or anatomically unique about facial lymphatics? Please discuss why the enhancer region's conservation is high in facial lymphatics.

   Significance

   • Describe the nature and significance of the advance (e.g. conceptual, technical, clinical) for the field.

   In this paper, the authors have revealed previously unknown regulatory sequences of zebrafish Prox1a. They identified enhancer sequences that control Prox1a expression in the facial collecting lymphatics and lymphatic valves. The loss of the -2.1prox1a enhancer led to malformation in some lymphatic valves, suggesting the functional importance of this enhancer.

   • State what audience might be interested in and influenced by the reported findings.

   Readers interested in vascular development and those interested in transcriptional control during development.

   • Define your field of expertise with a few keywords to help the authors contextualize your point of view. Indicate if there are any parts of the paper that you do not have sufficient expertise to evaluate.

   Lymphatic development in mammals, cardiac development, cardiology, cardiovascular pathology.

   Read the original source
4. 

   Review Commons

   Oct 27, 2023

   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

   Through the study of cis-regulatory element/enhancer activity in zebrafish, this study from Panara and colleagues provides insight into the transcriptional control of Prox1, a master regulator of lymphatic endothelial cell (LEC) fate. The authors used conservation of non-coding DNA, and chromatin accessibility data to identify enhancers that drive expression of a fluorescent reporter in anatomically distinct subsets of LECs. Analysis of transcription factor binding motifs in these enhancers suggests that differences in enhancer activity throughout the lymphatic vasculature may be due to binding of distinct transcription factors 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 #1

   Evidence, reproducibility and clarity

   Through the study of cis-regulatory element/enhancer activity in zebrafish, this study from Panara and colleagues provides insight into the transcriptional control of Prox1, a master regulator of lymphatic endothelial cell (LEC) fate. The authors used conservation of non-coding DNA, and chromatin accessibility data to identify enhancers that drive expression of a fluorescent reporter in anatomically distinct subsets of LECs. Analysis of transcription factor binding motifs in these enhancers suggests that differences in enhancer activity throughout the lymphatic vasculature may be due to binding of distinct transcription factors to these elements. Importantly, the authors identify a conserved 200 bp element within the -2.1kb enhancer that could drive expression in the lymphatic valve. Analysis of mutants carrying a 102 bp deletion in this region (including an Nfatc1 binding site), revealed reduced Prox1 expression and valve defects.

   Major comments

   • In the text it is suggested that sequence conservation was assessed across 8 species: "We aligned the region of the PROX1/prox1a locus in eight Osteichthyes species using mVISTA (Fig. 1A, Table S1)." Fig. 1A contains 7 species, and I am not able to find Table S1.
   • It would be important to discuss reasons that the +15.2kb enhancer is not clearly identifiable in the scATAC-seq analyses but drives expression. Is this due to the relatively limited activity in facial lymphatics? Furthermore, given the degree of conservation, it would be useful to mutate specific transcription factor binding motifs (e.g. Mafb, Sox18, etc) in the -15.2kb enhancer and assess activity in the FCLV.
   • OPTIONAL : Mutate Nr2f2 and Gata2 binding sites in -87kb enhancer to test for impact on activity. This would allow the authors to imply functional rather than sequence conservation. On a similar note, it would be interesting to understand if these enhancers are active in the context of mammalian lymphatic development.

   Minor comments:

   • It would be good to clarify in the following sentence that these enhancer marks are present at the whole embryo level and were not specifically identified in LECs : "Using zebrafish public databases for H3K4me1 and H3K27ac, we identified that ten of the selected prox1a CNEs were primed or active enhancers (Aday et al., 2011; Bogdanovic et al., 2012) (Fig. 1A, S1C)."
   • "....(Gupta et al., 2007) to determine the motifs and putative transcription factor binding sides." - should read "....(Gupta et al., 2007) to determine the motifs and putative transcription factor binding sites".
   • It might be more accurate to use zebrafish protein nomenclature for the transcription factor motifs in Fig1D, G and Fig2E (i.e. Gata2 not GATA2)

   Significance

   • While the roles of Prox1 in lymphatic vascular development and homeostasis are well established, relatively little is known about the cis-regulatory mechanisms governing it's expression; recent work has described an enhancer in the mouse Prox1 locus (Kazenwadel et al., 2023). This study from Panara and colleagues characterises numerous cis-regulatory elements in the zebrafish prox1a locus and demonstrates heterogeneous activity in anatomically distinct parts of the lymphatic vasculature. The imaging and characterisation of enhancer elements is of a high standard. Experiments that addressed the regulation of enhancers by upstream transcriptional or signalling cues would strengthen this study. Furthermore, to understand if there is functional conservation across evolution, analysis of activity in mouse embryos would be of interest.
   • This should be of broad interest to the vascular biology and developmental biology fields.
   • My expertise are in vascular biology, lymphatic development and developmental genetics.

   Read the original source
5. 

   Version published to 10.1101/2023.08.21.550483v1 on bioRxiv

   Aug 22, 2023