Characterization of the cellular components of mouse collecting lymphatic vessels reveals that lymphatic muscle cells are the innate pacemaker cells regulating lymphatic contractions

Reviewer #1 (Public review):

Summary:

This manuscript explores the multiple cell types present in the wall of murine collecting lymphatic vessels with the goal of identifying cells that initiate the autonomous action potentials and contractions needed to drive lymphatic pumping. Through the use of genetic models to delete individual genes or detect cytosolic calcium in specific cell types, the authors convincingly determine that lymphatic muscle cells are the origin of the action potential that triggers lymphatic contraction.

Strengths:

The experiments are rigorously performed, the data justify the conclusions and the limitations of the study are appropriately discussed.

There is a need to identify therapeutic targets to improve lymphatic contraction and this work helps identify lymphatic muscle cells as potential cellular targets for intervention.

Reviewer #2 (Public review):

Summary:

This is a well written manuscript describing studies directed at identifying the cell type responsible for pacemaking in murine collecting lymphatics. Using state-of-the-art approaches, the authors identified a number of different cell types in the wall of these lymphatics and then using targeted expression of Channel Rhodopsin and GCaMP, the authors convincingly demonstrate that only activation of lymphatic muscle cells produces coordinated lymphatic contraction and that only lymphatic muscle cells display pressure-dependent Ca2+ transients as would be expected of a pacemaker in these lymphatics.

Strengths:

The use of targeted expression of channel rhodopsin and GCaMP to test the hypothesis that lymphatic muscle cells serve as the pacemakers in musing lymphatic collecting vessels.

Weaknesses:

The only significant weakness was the lack of quantitative analysis of most of the imaging data shown in Figures 1-11. In particular, the colonization analysis should be extended to show cells not expected to demonstrate colocalization as a negative control for the colocalization analysis that the authors present. These weaknesses have been resolved by revision and addition of new and novel RNAseq data, additional colocalization data and membrane potential measurements.

Reviewer #3 (Public review):

Summary:

Zawieja et al. aimed to identify the pacemaker cells in the lymphatic collecting vessels. Authors have used various Cre-based expression systems and optogentic tools to identify these cells. Their findings suggest these cells are lymphatic muscle cells that drive the pacemaker activity in the lymphatic collecting vessels.

Strengths:

The authors have used multiple approaches to test their hypothesis. Some findings are presented as qualitative images, while some quantitative measurements are provided.

Weaknesses:

- More quantitative measurements.
- Possible mechanisms associated with the pacemaker activity.
- Membrane potential measurements.

Comments on revisions:

The authors have answered my comments with additional experiments, data and manuscript edits.

Author response:

The following is the authors’ response to the original reviews.

Public Reviews:

Reviewer #1 (Public Review):

Summary:

This manuscript explores the multiple cell types present in the wall of murine-collecting lymphatic vessels with the goal of identifying cells that initiate the autonomous action potentials and contractions needed to drive lymphatic pumping. Through the use of genetic models to delete individual genes or detect cytosolic calcium in specific cell types, the authors convincingly determine that lymphatic muscle cells are the origin of the action potential that triggers lymphatic contraction.

Strengths:

The experiments are rigorously performed, the data justify the conclusions, and the limitations of the study are appropriately discussed.

There is a need to identify therapeutic targets to improve lymphatic contraction and this work helps identify lymphatic muscle cells as potential cellular targets for intervention.

Weaknesses:

My only major comment would be that the manuscript provides a lot of rich information describing the cellular components of the muscular lymphatic vessel wall and that these data are not well represented by the title. The title (while currently accurate) could be tweaked to better represent all that is in this manuscript. Maybe something like

"Characterization/Interrogation of the cellular components of murine collecting lymphatic vessels reveals that lymphatic muscle cells are the innate pacemaker cells regulating lymphatic contractions" or "Discovery/Confirmation of lymphatic muscle cells as innate pacemaker cells of lymphatic contraction through characterization of the cellular components of murine collecting lymphatic vessels". Potentially a cartoon summary figure of the components that make up the collecting lymphatic vessel wall could also be included. In my opinion, these changes will make this manuscript of more interest to a broader group of scientists. I have a few additional comments for consideration to improve the clarity and enhance the discussion of this work.

We agree with the reviewer that our original manuscript, and our resubmission even more so with the addition of the scRNAseq data, provides a significant amount of information regarding the composition of the lymphatic collecting vessel wall. We have changed our title to match one suggestion of the reviewer: “Characterization of the cellular components of murine collecting lymphatic vessels reveals that lymphatic muscle cells are the innate pacemaker cells regulating lymphatic contractions".

Reviewer #2 (Public Review):

Summary:

This is a well-written manuscript describing studies directed at identifying the cell type responsible for pacemaking in murine-collecting lymphatics. Using state-of-the-art approaches, the authors identified a number of different cell types in the wall of these lymphatics and then using targeted expression of Channel Rhodopsin and GCaMP, the authors convincingly demonstrate that only activation of lymphatic muscle cells produces coordinated lymphatic contraction and that only lymphatic muscle cells display pressure-dependent Ca2+ transients as would be expected of a pacemaker in these lymphatics.

Strengths:

The use of a targeted expression of channel rhodopsin and GCaMP to test the hypothesis that lymphatic muscle cells serve as the pacemakers in musing lymphatic collecting vessels.

Weaknesses:

The only significant weakness was the lack of quantitative analysis of most of the imaging data shown in Figures 1-11. In particular, the colonization analysis should be extended to show cells not expected to demonstrate colocalization as a negative control for the colocalization analysis that the authors present.

We understand the reviewer’s concern regarding the lack of a control for the colocalization analysis and that the colocalization analysis was limited to just one set of cell markers. We have now provided a colocalization analysis of Myh11 and PDGFRα, to serve as a co-localization negative control based on our RT-PCR and scRNASeq findings, which is incorporated into the current Supplemental figure 1. In regard to the staining pattern of other various marker combinations, the results were often quite clear with the representative images that two separate cell populations were being stained such as the case with labeling endothelial cells with CD31, macrophage labeling with the MacGreen mice, or hematopoietic cells with CD45.

During our lengthy rebuttal process we completed a single cell RNA sequence analysis using our isolated and cleaned mouse inguinal axillary lymphatic collecting vessels to aid in our characterization of the vessel wall and to more thoroughly answer these questions regarding colocalization in arguably a robust manner. The generation of our scRNAseq dataset, derived from isolated and cleaned mouse inguinal axillary collecting vessels from 10 mice, 5 male and 5 females, allowed us to profile over 2200 of the adventitial fibroblast like cells (AdvCs) we had identified in our original submission. Using this dataset, we were able to confirm co-expression of Cd34 and Pdgfrα in AdvCs and assess the co-expression of other genes of interest from our RT-PCR experiments and immunofluorescence experiments. This approach will also allow other lymphatic investigators to assess their genes of interest as our dataset is uploaded to the NIH Gene Omnibus and will be uploaded to the Broad Institute Single Cell Portal upon publication.

Here we show that the vast majority of non-muscle fibroblast like cells referred to as AdvCs were double positive for both CD34 and PDGFRα. We also show that the AdvCs that express commonly used pericyte markers Pdgfrb and Cspg4 also co-expressed Pdgfrα. Critically, this data also shows that the AdvCs that express genes linked with lymphatic contractile dysfunction (Ano1, Gjc1 or connexin 45, and Cacna1c “Cav1.2”) co-express Pdgfrα and would render these genes susceptible to Cre-mediated recombination using our Pdgfrα-CreER^TM model.

Reviewer #3 (Public Review):

Summary:

Zawieja et al. aimed to identify the pacemaker cells in the lymphatic collecting vessels. Authors have used various Cre-based expression systems and optogenetic tools to identify these cells. Their findings suggest these cells are lymphatic muscle cells that drive the pacemaker activity in the lymphatic collecting vessels.

Strengths:

The authors have used multiple approaches to test their hypothesis. Some findings are presented as qualitative images, while some quantitative measurements are provided.

Weaknesses:

- More quantitative measurements.

- Possible mechanisms associated with the pacemaker activity.

- Membrane potential measurements.

We thank the reviewers for their concerns and have addressed them in the following manner.

- We added novel single cell RNA sequencing of isolated and cleaned inguinal axillary vessels from 10 mice (5 males and 5 females). This allowed us to quantify the number of AdvCs that coexpress CD34 and Pdgfrα as well as the number of cells co-expressing Pdgfrα and other markers.

- We have added a negative control with quantification for the co-localization analysis assessing Myh11 and Pdgfrα. We have added a negative control with quantification for the ChR2-photo stimulated contraction experiments using Myh11CreERT2-ChR2 mice that were not injected with tamoxifen.

- We also used Biocytin-AF488 in our intracellular Vm electrodes to map the specific cells in which we recorded action potentials and in neighboring cells since Biocytin-AF488 is under 1KDa and can pass through gap junctions. This approach independently labeled lymphatic muscle cells and their direct neighbors for 3 IALVs from 3 separate mice.

- We performed membrane potential recordings in isolated, pressurized (under isobaric conditions), and spontaneously contracting inguinal axillary lymphatic collecting vessels at different pressures.

- We also show that the pressure-frequency relationship is dependent on the slope of the diastolic depolarization as no other parameter was significantly altered in our study and the diastolic depolarization slope was highly correlated with contraction frequency.

We believe the addition of these novel data, controls, experiments, and quantifications have improved the manuscript in line with the reviewers’ suggestions.

Recommendations for the authors:

Reviewer #1 (Recommendations For The Authors):

Lines 149-162: The authors rule out the methylene blue staining cells in the cLV wall as pacemakers because they don't form continuous longitudinal connections to drive propagation. Is it possible for a pacemaker cell to only initiate the contraction and then have the LMCs make the axial electrical connections to propagate the electrical wave? I am not trying to suggest the methylene blue cells are pacemakers, but I am not sure the lack of longitudinal (or radial) connectivity is sufficient evidence to rule out the possibility. This comment also is relevant to the 3 criteria for a pacemaker cell listed in the Discussion (Lines 413-417).

We agree with the reviewer’s broader point that a pacemaker cell may not require direct contact with other ‘pacemaker’ cells within the tissue as long as they are still within the same electrical syncytium. However, we do expect a continuous presence of a pacemaker cell type throughout the vessel wall length to account for the persistence of spontaneous contractile behavior despite vessel length, and the ability for contraction initiation to shift (Akl et al 2011, Castorena et al 2018 and Castorena et al 2022) and the occurrence of spontaneous action potentials. In Dirk van Helden’s seminal work in 1993 on lymphatic pacemaking, a major finding was that “SM of small lymphangions or that of short segments, cut from lymphangions of any length, behaved similarly”. We have adjusted our phrase regarding the requirement of a contiguous network and instead suggest a continuous presence along the vessel network and integrated into the electrical syncytium.

Methylene blue is an alkaline stain that will stain acidic structures and historically methylene blue is noted to stain Interstitial cells of Cajal in the gastrointestinal tract which typically exist as network of cells(Huizinga et al 1993 and Berezin 1988). No such network was readily apparent in our methylene blue staining nor did the stained cells have a similar morphology to the ICCs of the gastrointestinal tract. Further, methylene blue is staining is not limited to ICCs or pacemaker cells at large as it has been used to kill cancer cells. Within the small intestine methylene blue was noted to also stain macrophage like cells (Mikkelsen et al 1988), and we too draw parallels between the macrophage morphology observed with Macgreen mice and methylene-blue stained cells. The specific structure for the ICC affinity for methylene blue is not well described and while the innate cytotoxicity of methylene blue and light has been used to kill ICCs and impair slow wave generation, the lack of specificity of this method leaves much to be desired. What is clear is that the ICC network highlighted by methylene blue in the gut is absent in lymphatic collecting vessels.

In Figure 15/Video12, is it possible that the cells that are showing intracellular Ca2+ in diastole are the cells that reach a threshold membrane potential that then trigger the rest of the LMCs? As the authors have shown heterogeneity in the LMCs surface markers, is it possible that the cells with Ca2+ activity during diastole are identifiable by a distinct molecular phenotype? Or is the thought that these cells are randomly active in diastole? Some discussion/speculation about this seems appropriate.

We are in agreement with the reviewer’s conclusion that there is heterogeneity in the LMCs as it pertains to the calcium oscillations in diastole, either under normal buffer conditions or when L-type channels are inhibited with nifedipine. We also note significant heterogeneity in the gene expression noted within the four LMC subclusters (0-3), though we did not see significant differences in either in Ip3R1 or Ano1 expression. However, subcluster “0” had increased expression of Itprid2, also known as KRas-induced actin-interacting protein (KRAP) which is thought to tether, and thus immobilize, IP3 receptors to the actin cortex beneath the cell membrane. KRAP has been recently proposed to be a critical player in IP3 receptor “licensing” which allows IP3 receptors to release calcium (Vorontsova et al., 2022). However, whether similar requirement of IP3R licensing is necessitated in all cells or specifically in LMCs is unknown it is quite clear there are specific release sites within the cell and this topic is currently under further investigation for a separate manuscript. We would like to note that there is yet to be a clear consensus on whether IP3R licensing is required as much of these studies are performed in cultured cells and this mechanism has only recently been described.

Healthy lymphatic collecting vessels typically have a single pacemaker driving a coordinated propagated contraction in ex vivo isobaric myograph studies (Castorena-Gonzalez et al., 2018), which is typically at either end of the cannulated vessel. We believe that this is due to the lack of a bordering cell in one direction and allows charge to accumulate and voltage to reach threshold at these sites preferentially. We have tried to image calcium at the pacemaking pole of the vessel to observe the specific Ca²⁺ transients at these sites though invariably the act of imaging GCaMP6f results in the pacemaker activity initiating from the other pole of the vessel. It is our opinion that the fact that LMCs are heterogenous in their Ca²⁺ transients is a feature to the system as it permits a wider range of depolarization signals, and thus allows finer control of the pacing as different physical/pressure or signaling stimuli is encountered. Likely, the cells with the higher propensity of Ca²⁺ transients act as the contraction initiation site in vivo, though it must also be noted that the LMC density decreases around lymphatic valve sites. In fact, in guinea pig collecting vessels there are very few LMCs at the valves which can render them electrically uncoupled or poorly coupled (Van Helden, 1993). Thus, valve sites in which there is greater electrical resistance due to lower LMC-LMC coupling may allow for charge accumulation in the LMCs at the valve site, similar to the artificial condition achieved in our myograph preparations with two cut ends, and allow them to reach threshold first and drive coordination at the valve sties.

An additional description of what the PTCL analysis is meant to represent physiologically would be helpful for readers.

We have better described the conversion of the calcium signals into “particles” for analysis at first mention in the methods and results section and have included the requisite reference to this specific methodology in Line 429-30.

A description of how DMAX is experimentally determined is needed.

We have adjusted our methods section to describe DMAX in line 774-775.

“with Ca²⁺-free Krebs buffer (3mM EGTA) and diameter at each pressure recorded under passive conditions (DMAX).”

I think the vessels referred to as popliteal lymphatic vessels are actually saphenous lymphatic vessels (afferent to the popliteal lymph node). Please clarify.

Indeed, some of the vessels used in this study are the afferents to the single popliteal node. They travel with the caudal branch of the saphenous vein, but have routinely been described as popliteal vessels, as opposed to saphenous lymphatic vessels, by the lymphatic field at large (Tilney 1971 PMCID: PMC1270981, Liao 2015 PMID: 25512945). To move away from this nomenclature would likely add to confusion although we agree that the lymphatic field may need to improve or correct the vessel naming paradigm to match the vascular pairs they follow.

Reviewer #2 (Recommendations For The Authors):

Lines 214-215 - can you cite a reference for the observation that rhythmic contractions don't require the presence of valves?

We have added the reference. In Dr. Van Helden’s seminal work on the topic in 1993, “Vessel segments were then cut from selected small lymphangions (length 300-500 um) by cutting at the valves.” Additionally, work by Dr Anatoliy Gashev utilized sections of lymphatic vessels that lacked valves to study orthograde and retrograde shear sensitivity (Gashev et al., 2002).

Lines 224-230 - It would have been nice to see colocalization analysis for all cell types so that "negative" results could be compared with the "positives" that you report. This would help bolster evidence of your ability to separate cell types.

We understand the reviewer’s sentiment and agree. We have now added a “negative control” colocalization staining and analysis for PDGFR and Myh11 which has been added to the current SuppFigure 1. We stained 3 IALVs from 3 separate mice with PDGFRα and Myh11 and performed confocal microscopy. We ran the FIJI BIOP-JACOP colocalization plugin as before and observed very little colocalization of the two signals. Additionally, we have also added a coexpression assessment for CD34 and PDGFRα and other genes using our scRNAseq dataset.

line 293 - Should read "Cx45 in..."

This has been corrected.

“The expression of the genes critically involved in cLV function—Cav1.2, Ano1, and Cx45—in the PdgfrαCreER^TM-ROSA26mTmG purified cells and scRNAseq data prompted us to generate PdgfrαCreER^TM-Ano1^fl/fl, PdgfrαCreER^TM-Cx45^fl/fl, and PdgfrαCreER^TM-Cav1.2^fl/fl mice for contractile tests.”

lines 470-473 - A reference for this statement should be cited.

We have added the reference. In Dr. Van Helden’s seminal work on the topic in 1993, “Vessel segments were then cut from selected small lymphangions (length 300-500 um) by cutting at the valves.” Additionally, work by Dr Anatoliy Gashev utilized sections of lymphatic vessels that lacked valves to study orthograde and retrograde shear sensitivity (Gashev et al., 2002).

Lines 483-487 - References should be cited for these statements.

We have narrowed and clarified this statement and supported it with the necessary citations.

“Of course, mesenchymal stromal cells (Andrzejewska et al., 2019) and fibroblasts (Muhl et al., 2020; Buechler et al., 2021; Forte et al., 2022) are present, and it remains controversial to what extent telocytes are distinct from or are components/subtypes of either cell type (Clayton et al., 2022). Telocytes are not monolithic in their expression patterns, displaying both organ directed transcriptional patterns as well as intra-organ heterogeneity (Lendahl et al., 2022) as readily demonstrated by recent single cell RNA sequencing studies that provided immense detail about the subtypes and activation spectrum within these cells and their plasticity (Luo et al., 2022).”

Lines 584-585 - Missing a reference citation.

Thank you for catching this error, the correct citation was for Boedtkjer et al 2013 and is now properly cited.

Line 638 - "these this" should read "this"

Thank you for catching this error. This particular sentence was removed in light of the addition of the scRNAseq data.

Reviewer #3 (Recommendations For The Authors):

This manuscript from Zawieja et al. explored an interesting hypothesis about the pacemaker cells in lymphatic collecting vessels. Many aspects of lymphatic collecting vessels are still under investigation; hence this work provides timely knowledge about the lymphatic muscle cells as a pacemaker. Although it is an important topic of the investigation, the data provided do not support the overall goal of the manuscript. Many figures (Figure 1-5) provide quantitative estimation and the description provided in the results section might only be useful for a restricted audience, but not to the broader audience. Some of the figures are very condensed with multiple imaging panels and it is hard to follow the differences in qualitative analysis. Overall, this manuscript can be improved by more streamlined description/writing and figure arrangements (some of the figures/panels can be moved to the supplementary figures).

We disagree with the notion that the original data provided did not support the goal of the manuscript- to identify and test putative pacemaker cell types. Nonetheless we believe we have also added ample novel data to the manuscript, including membrane potential recordings and scRNAseq to highlight and to add further support to our conclusion that the pacemaker cell is an LMC. We believe the scRNAseq data will also greatly enhance the appeal of the manuscript to a broader audience and have renamed the manuscript in line with the wealth of data we have collected on the components of the vessel wall as we tested for putative pacemaker cells.

As requested, we have moved many figures to the supplement to allow readers to focus more on the more critical experiments.

A few other points that need to be addressed:

(1) Authors used immunofluorescence-based differences in various cell types in the collecting vessels. Initially, they chose ICLC, pericytes, and lymphatic muscle cells. But then they started following adventitial cells and endothelial cells. It is not clear from the description, why these other cells could be possibly involved in the pacemaker activity. It will be easier to follow if authors provide a graphical abstract or summary figure about their hypothesis and what is known from their and others' work.

We would like to clarify that we used the endothelial cells as controls to ensure what we observed via immunofluorescence and FACs RT-PCR were a separate cell type from either lymphatic muscle or lymphatic endothelial cells on the vessel wall. Staining for the endothelium also allowed us to assess where these PDGFRα+CD34+ cells reside in the vessel wall. We started with a wide range of markers that are conventionally used for targeting specific cell types, but as expected those markers are not always 100% specific. Specifically, we focused on CD34, Kit, and Vimentin as those were the markers for the non-muscle cells observed in the lymphatic collecting vessel wall previously. What we found was that CD34 and PDGFRα labeled the same cell type. As there was not a CD34Cre mouse available at the time we instead utilized the inducible PDGFRαCreERTM. We are unsure how well an abstract figure will condense the conclusions from the experiments listed here but if absolutely required for publication we can attempt to highlight the representative cell populations identified on the vessel wall.

(2) Authors used many acronyms in the manuscript without defining them (when they appeared for the first time). Please follow the convention.

We have checked the manuscript and made several corrections regarding the use of abbreviations.

(3) How specific PDGFR-alpha as a marker of the pericytes? It can also label the mesenchymal cells. Why did the author choose PDGFR-alpha over beta for their Cre-based expression approach?

We tried to assess if there were a pericyte like cell present in or along the wall using PDGFRbeta (Pdgfrβ). Pdgfrβ is commonly used to identify pericytes (Winkler et al., 2010), while in contrast Pdgfrα is a known fibroblast marker (Lendahl et al., 2022). Pdgfrβ CreERT2 resulted in recombination in both LMCs and AdvCs, preventing it from being a discriminating marker for our study where as Myh11CreER^T2 and PDGFRαCreER^TM were specific at least to cell type based on our FACSs-RT-PCR and staining. As you can tell from the scRNAseq data in Figure 5, there was no cell cluster that Pdgfrβ was specific for in contrast to PDGFRα and Myh11. In Figure 6 we show the expression of another commonly used pericyte marker NG2 (Cspg4) in our scRNAseq dataset which was observed in both LMCs and AdvCs as well. Lastly, MCAM (Figure 6) can also be a marker for pericytes though we see only expression in the LMCs and LECs for this marker. Notably, almost all of the AdvCs express PDGFRα rendering the PDGFRαCreER^TM a powerful tool to study this population of cells on the vessel wall including those that were PDGFRα+Cspg4+ or PDGFRα+ Pdgfrβ+.

We were reliant on PDGFRαCreER^TM as that was the only available PDGFRα Cre model at the time. Note we used PdgfrβCreER^T2 and Ng2Cre in our study but found that both Cre models recombined both LMCs and AdvCs.

(4) Please include appropriate references for all the labeling markers (PDGFR-alpha, beta, and myc11 etc.) that are used in this manuscript.

We have added multiple references to the manuscript to support the use of these common cell “specific” markers as of course each marker is limited in some capacity to fully or specifically label a single population of cells (Muhl et al., 2020).

(5) One of the criteria for the pacemaker cells is depolarization-induced propagated contractions. Authors have used optogenetics-induced depolarization to test this phenomenon. Please include negative controls for these experiments.

We have now added negative controls to this experiment which were non-induced (no tamoxifen) Myh11CreER^T2-Chr2 popliteal vessels. This data has been added to the Figure 8.

(6) What are the resting membrane potentials of Lymphatic muscle cells? The authors should provide some details about this in the manuscript.

We agree with the reviewer and have added membrane potential recordings (Figure 13) at different pressures and filled our recording electrode with the cell labeling molecule BiocytinAF488 to highlight the action potential exhibiting cells, which were the LMCs. Lymphatic resting membrane potential is dynamic in pressurized vessels, which appears to be a critical difference in this approach as compared to pinned out vessels or those on wire myographs likely due to improper stretch or damage to the vessel wall. In mesenteric lymphatic vessels isolated from rats the minimum membrane potential achieved during repolarization ranges from -45 to 50mV typically while IALVs from mice are typically around -40mV, though IALVs have a notably higher contraction frequency. Critically, we have also added novel membrane potential recordings to this manuscript in IALVs at different pressures and show that the diastolic depolarization rate is the critical factor driving the pressure-dependent frequency.

(7) In the discussion, the authors discussed SR Ca2+ cycling in Pacemaking, but the relevant data are not included in this manuscript, but a manuscript from JGP (in revision) is cross-referenced.

As discussed above, we have recently published our work where studied IALVs from Myh11CreERT2-Ip3R1fl/fl (Ip3r1ismKO) and Myh1CreERT2-Ip3r1fl/fl-Ip3r2fl/fl-Ip3r3fl/fl mice (Zawieja et al., 2023). Deletion of Ip3r1 from LMCs recapitulated the dramatic reduction in frequency we previously published in Myh11CreERT2-Ano1fl/fl mice and the loss of pressure dependent chronotropy. Furthermore, in this manuscript we also showed that the diastolic calcium transients are nearly completely lost in ILAVs from Myh11CreERT2-Ip3R1fl/fl knockout mice. There was no difference in the contractile function between IALVs from single Ip3r1 knockout and the triple Ip3r1-3 knockout mice suggesting that it is Ip3r1 that is required for the diastolic calcium oscillations. Further, in the presence of 1uM nifedipine there were still no calcium oscillations in the Myh11CreERT2-Ip3r1fl/fl LMCs. These findings provide further support for our interpretation that the pacemaking is of myogenic origin.

Andrzejewska, A., B. Lukomska, and M. Janowski. 2019. Concise Review: Mesenchymal Stem Cells: From Roots to Boost. Stem Cells. 37:855-864.

Buechler, M.B., R.N. Pradhan, A.T. Krishnamurty, C. Cox, A.K. Calviello, A.W. Wang, Y.A. Yang, L.

Tam, R. Caothien, M. Roose-Girma, Z. Modrusan, J.R. Arron, R. Bourgon, S. Muller, and S.J. Turley. 2021. Cross-tissue organization of the fibroblast lineage. Nature. 593:575579.

Castorena-Gonzalez, J.A., S.D. Zawieja, M. Li, R.S. Srinivasan, A.M. Simon, C. de Wit, R. de la Torre, L.A. Martinez-Lemus, G.W. Hennig, and M.J. Davis. 2018. Mechanisms of Connexin-Related Lymphedema. Circ Res. 123:964-985.

Clayton, D.R., W.G. Ruiz, M.G. Dalghi, N. Montalbetti, M.D. Carattino, and G. Apodaca. 2022. Studies of ultrastructure, gene expression, and marker analysis reveal that mouse bladder PDGFRA(+) interstitial cells are fibroblasts. Am J Physiol Renal Physiol. 323:F299F321.

Forte, E., M. Ramialison, H.T. Nim, M. Mara, J.Y. Li, R. Cohn, S.L. Daigle, S. Boyd, E.G. Stanley, A.G. Elefanty, J.T. Hinson, M.W. Costa, N.A. Rosenthal, and M.B. Furtado. 2022. Adult mouse fibroblasts retain organ-specific transcriptomic identity. Elife. 11.

Gashev, A.A., M.J. Davis, and D.C. Zawieja. 2002. Inhibition of the active lymph pump by flow in rat mesenteric lymphatics and thoracic duct. J Physiol. 540:1023-1037.

Lendahl, U., L. Muhl, and C. Betsholtz. 2022. Identification, discrimination and heterogeneity of fibroblasts. Nat Commun. 13:3409.

Luo, H., X. Xia, L.B. Huang, H. An, M. Cao, G.D. Kim, H.N. Chen, W.H. Zhang, Y. Shu, X. Kong, Z.

Ren, P.H. Li, Y. Liu, H. Tang, R. Sun, C. Li, B. Bai, W. Jia, Y. Liu, W. Zhang, L. Yang, Y. Peng, L. Dai, H. Hu, Y. Jiang, Y. Hu, J. Zhu, H. Jiang, Z. Li, C. Caulin, J. Park, and H. Xu. 2022. Pancancer single-cell analysis reveals the heterogeneity and plasticity of cancer-associated fibroblasts in the tumor microenvironment. Nat Commun. 13:6619.

Muhl, L., G. Genove, S. Leptidis, J. Liu, L. He, G. Mocci, Y. Sun, S. Gustafsson, B. Buyandelger, I.V.

Chivukula, A. Segerstolpe, E. Raschperger, E.M. Hansson, J.L.M. Bjorkegren, X.R. Peng, M. Vanlandewijck, U. Lendahl, and C. Betsholtz. 2020. Single-cell analysis uncovers fibroblast heterogeneity and criteria for fibroblast and mural cell identification and discrimination. Nat Commun. 11:3953.

Van Helden, D.F. 1993. Pacemaker potentials in lymphatic smooth muscle of the guinea-pig mesentery. J Physiol. 471:465-479.

Vorontsova, I., J.T. Lock, and I. Parker. 2022. KRAP is required for diffuse and punctate IP(3)mediated Ca(2+) liberation and determines the number of functional IP(3)R channels within clusters. Cell Calcium. 107:102638.

Winkler, E.A., R.D. Bell, and B.V. Zlokovic. 2010. Pericyte-specific expression of PDGF beta receptor in mouse models with normal and deficient PDGF beta receptor signaling. Mol Neurodegener. 5:32.

Zawieja, S.D., G.A. Pea, S.E. Broyhill, A. Patro, K.H. Bromert, M. Li, C.E. Norton, J.A. CastorenaGonzalez, E.J. Hancock, C.D. Bertram, and M.J. Davis. 2023. IP3R1 underlies diastolic ANO1 activation and pressure-dependent chronotropy in lymphatic collecting vessels. J Gen Physiol. 155.