Lymphatic muscle cells are the innate pacemaker cells regulating mouse lymphatic collecting vessel 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.

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.

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.

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.