Cell Biology

Cellular characterization of the mouse collecting lymphatic vessels reveals that lymphatic muscle cells are the innate pacemaker cells

Scott D Zawieja author has email address
Grace A Pea
Sarah E Broyhill
Advaya Patro
Karen H Bromert
Charles E Norton
Hae J Kim
Sathesh K Sivasankaran
Min Li
Jorge A Castorena-Gonzalez
Bernard T Drumm
Michael J Davis author has email address

Department of Medical Pharmacology & Physiology, University of Missouri, Columbia, United States
Bioinformatics and Analytics Core, Division of Research, Innovation and Impact, University of Missouri, Columbia, United States
Department of Pharmacology, Tulane University, New Orleans, United States
Smooth Muscle Research Centre, Dundalk Institute of Technology, Dundalk, Ireland

https://doi.org/10.7554/eLife.90679.3

Open access
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Figures and data

Methylene blue staining of isolated mouse IALVs
Representative image of an isolated and cleaned IALV after methylene blue staining which revealed cells of various morphology. (B) is the zoomed in image of the yellow dotted box in A which contained large ovoid cells with granular staining (B, yellow asterisks). Fine cellular extensions (red asterisks) stained by methylene blue in some cells were visualized with color channel separation and division (C). (D, E) Similar as B and C, but in a separate vessel which stained with a higher density of methylene blue stained cells some of which had limited cellular processes. F) Focal reconstruction from imaging a methylene blue stained IALV using an upright microscope and immersion objective.

Staining Mouse IALVs for ICLC Markers
Representative immunofluorescent max projections of half vessel confocal image stacks imaged from mouse IALVs stained for ICLC markers. DAPI (A), cKIT (B), and CD34 (C) and their merged image (D). Representative max projections of the intermediate filament VIMENTIN (E), the intermediate filament desmin (F), CD34 (G) and their merged image (H). Representative max projection of VIMENTIN (I), cKIT (J), CD34 (K) and their merged image (L). Scale bar = 100 µm for all images.

Immunofluorescence Labeling of Mouse IALVs with Markers for ICLC, LMC, LEC, and Immune Cell Populations
We stained isolated mouse IALVs with cellular markers used to differentiate various cell types observed in cLVs. Half vessel image stacks were taken with confocal microscopy and the resulting representative max projections are shown. (A) CD34 stained cells and LMC staining with SMA (B) and calponin (C) and the corresponding merged (D) image. There was significant overlap in (E) CD34 staining along with the fibroblast marker PDGFRα compared to LMC staining with SMA (G) and the merged (H) image. The endothelial marker CD31 (I) to delineate LECs with PDGFRα staining (J), and the LMC marker calponin (K) with the merged image (L) revealed 3 separate populations of cells. PDGFRβ (O) stained many cells that were CD34 (M) and PDGFRα (N) positive, as seen in the merge imaged (P), in addition to PDGFRβ signal detected in the LMC layer (Q). Max projections of only the luminal frames of a z-stack at lymphatic valve locations revealed PDGFRβ, CD34, and PDGFRα labeling in bipolar shaped cells with long extensions that traveled throughout the valve leaflets (V, W). d Control IALV (Y) stained only with secondary antibody. Scale bar = 100 µm for all images.

iCre-ROSA26mTmG Labelling and Fidelity to Target Putative Pacemaker Cell Populations
Stitched montages of serial max projections of GFP and tdTomato signal from live IALVs isolated from PdgfrαCre-ROSA26mTmG (A), Ng2Cre-ROSA26mTmG (B), PdgfrαCreER^TM-ROSA26mTmG (C), PdgfrβCreER^T2-ROSA26mTmG (D), cKitCreER^T2-ROSA26mTmG (E), and Myh11CreER^T2- ROSA26mTmG (F). IALVs were digested into single cells and GFP⁺ cells were purified via FACS from Prox1-eGFP (G), Myh11CreER^T2-ROSA26mTmG (H), PdgfrαCreER^TM-ROSA26mTmG (I), and PdgfrβCreER^T2-ROSA26mTmG (J) mice. Representative gels demonstrating RT-PCR products corresponding to the respective genes used in the promoter of each specific transgene employed to drive either eGFP or Cre mediated recombination of ROSA26mTmG from each GFP⁺ sorted population (K-N) to assess fidelity. Images are representative of IALVs from at least 3 separate mice. FACs and RT-PCR was repeated at least 3 times for each mouse.

scRNAseq analysis of mouse IALVs from ROSA26mTmG mice.
IALVs were cleaned and isolated from 8 ROSA26mTmG mice and digested into a single cell suspension for scRNAseq analysis with the 10X platform. A) UMAP of the various cell populations that compromise the mouse IALV though some mammary epithelia contamination was present (populations 18,19). B) Heat map of commonly used genes for cell identification for each of the cell clusters. Dot plots to assess cell cluster expression of the genes shown in Figure 4 using a dot plot for the LEC markers Prox1 (C) and Flt4 (D, VEGFR3), LMC markers Myh11 (E) and caponin1 (F, Cnn1), fibroblast markers Pdgfrα (G) and Lum (H, Lumican), ICC marker Kit (I), the pericyte and smooth muscle precursor marker (Pdgfrβ), and the hematopoietic marker Ptprc (K, CD45).

RT-PCR Profiling of FACs Purified Cells from iCre-ROSA26mTmG
Expanded RT-PCR profiling of genes to discriminate LECs, LMCs, and other cell types in our GFP⁺ sorted cells from Prox1-eGFP (A), Myh11CreER^T2-ROSA26mTmG (B), PdgfrβCreER^T2-ROSA26mTmG (C), and PdgfrαCreER^TM-ROSA26mTmG (D). Dot plots for the genes assessed in A-D in our IALV scRNAseq analysis confirmed those results. In addition to a population of AdvCs expressing Cacna1c, we also noted expression of Cx45 (N) which was also observed in LECs) and Ano1 (O) in the AdvC clusters. We confirmed this expression using GFP⁺ cells sorted from PdgfrαCreER^TM-ROSA26mTmG IALVs for RT-PCR (P) and ruled out hematopoietic or LEC contamination. All RT-PCRs were performed 2-4 times for each gene over each sorted cell population collected from different mice.

Isobaric contractile Assessment of popliteal cLV from PdgfrαCreER^TM driven deletion of Ano1, CX45, and CaV1.2
Summary of the contractile parameters recorded from popliteal cLVs in PdgfrαCreER^TM-Ano1^fl/fl, PdgfrαCreER^TM-Cx45^fl/fl mice, PdgfrαCreER^TM-Cav1.2^fl/fl mice. Contraction frequency (A, D, G), ejection fraction (B, E, H), and vessel tone (C, F, I) were assessed. No statically significant differences observed in cLVs isolated from PdgfrαCreER^TM-Ano1^fl/fl and PdgfrαCreER^TM-Cx45^fl/fl mice across these three parameters. Mean and SEM shown, n=6 popliteal vessels from 3 mice PdgfrαCreER^TM-Ano1^fl/fl mice and n=10 popliteal vessels from 6 mice Ano1^fl/fl mice. Mean and SEM shown, n=5 popliteal vessels from 3 mice PdgfrαCreER^TM-CX45^fl/fl mice and n=8 popliteal vessels from 11 mice CX45^fl/fl mice. Mean and SEM shown, n=6 popliteal vessels from 3 mice PdgfrαCreER^TM-Cav1.2^fl/fl mice and n=9 popliteal vessels from 20 mice Cav1.2^fl/fl mice. The contractile data from control Cav1.2^fl/fl vessels was previously published but was separated by sex (Davis et al., 2022) while they are combined here. * Denotes significance at p <0.05 which 0.10 > p >0.05 are reported as text. Normalized contraction amplitude, fractional pump flow, end diastolic diameter can be found in SuppFigure 8.

ChR2-Mediated Depolarization Only in LMCs Triggers Contraction
Representative max projections of tdTomato-ChR2 signal in popliteal cLVs isolated from cKitCreER^T2- ChR2-tdTomato (A), PdgfrαCreER^TM-ChR2-tdTomato (C), and Myh11CreER^T2- ChR2-tdTomato (E) with their corresponding brightfield image (B, D, F) respectively. Time-lapse brightfield images every 0.5 s starting at stimulation t=0 for cKitCreER^T2-ChR2-tdTomato (G-J), PdgfrαCreER^TM-ChR2-tdTomato (K- N), and Myh11CreER^T2- ChR2-tdTomato (O-R). The I bar denotes the inner diameter at t=0 over time and white asterisks denote the contraction. Representative diameter trace for the popliteal cLV demonstrate spontaneous contractions with the dotted boxes indicating the optical stimulation event in the respective brightfield images of the time lapse images. Isolated cLVs from cKitCreER^T2-ChR2-tdTomato (S), PdgfrαCreER^TM-ChR2-tdTomato (T), and Myh11CreER^T2- ChR2-tdTomato (U) were stimulated with light pulses (red dashed lines) and the summation of contraction triggering for each genotype (V). Mean and SEM are shown, **** denotes p<0.0001. Contraction recorded from at least 6 popliteal cLVs from 3 mice per genotype.

cKitCreERT2 Drives GCaMP6f Expression Primarily in Mast Cells in Mouse IALVs
Representative max projection of GCaMP6f signal over time in an IALV isolated from a cKitCreER^T2- GCaMP6f mouse with ROI indicated around individual cells, primarily large ovoid cells, but also including a circumferential LMC (Cell10) and a horizontal LEC (Cell 11). Of cells 1-9, only cell 7 had any Ca²⁺ activity (red arrows) during the recording time as indicated by the STMs from each ROI (B) and their normalized F/F₀ plots in (C). In contrast, the LMC in ROI 10 had both rhythmic global Ca²⁺ events (D) that spanned the cell axis (vertical axis) in the STM (E) in addition to localized Ca²⁺ events intervening the time between global events (green arrows). Representative max projection of GCaMP6f signal over time after stimulation with C48-80 (F) with many large ovoid cells displaying long lasting global Ca²⁺ events (G, H) while not immediately affecting the LMC Ca²⁺ dynamics (I, J).

Lack of coordinated Ca²⁺ Activity Across Contraction Cycle in PDGFRα Cells
Representative max projections of GCaMP6f signal over time in an IALVs isolated from PdgfrαCreER^TM- GCaMP6f mice (A, D). ROIs were made around cells and GCaMP6f recorded over time to generate the corresponding STMs (B, E) for each cell and plots (C, F) respectively. Once again, incidental recombination occurred in a LMC which displayed rhythmic Ca²⁺ flashes (C) while the slight undulation in the other cells is due to movement artifact (B). Red arrows indicate the limited local Ca²⁺ activity observed in two cells from a PdgfrαCreER^TM-GCaMP6f IALV.

Heterogeneous Diastolic Ca²⁺ Transient Activity in LMCs
Representative max projections of GCaMP6f signal over time in an IALVs isolated from Myh11CreER^T2-GCaMP6f mice (A). LMCs were outlined with ROIs to assess GCaMp6F signal over time. Rhythmic global flashes (B) were entrained across all the LMCs in the FOV (C) with many cells exhibiting diastolic Ca²⁺ release events. Cells exhibiting at least one diastolic Ca²⁺ event, within the context of our focal plane constraints, over the recorded time were denoted by the red asterisks. The plot in (D) magnifies the first diastolic period, seconds 1-3 of C to assist in visualizing the lack of coordination of the diastolic events. (E) Max projection of the pseudo-linescan analysis across the axis of the vessel to highlight diastolic Ca²⁺ transients in all cells in the field of view and their lack of coordination across the cells (x-axis). The white dotted box shows the first diastolic period plotted in (D).

Pressure Dependency of Mouse LMC Diastolic Ca²⁺ Transients
Representative max projection of GCaMP6f signal over 20 s in an IALVs isolated from Myh11CreER^T2-GCaMP6f mice in the presence of the L-type blocker nifedipine (1μM) (A) pressurized to 0.5 cmH₂O, 2 cmH₂O, 5 cmH₂O. The local diastolic Ca²⁺ transients persist in the presence of nifedipine and increase with increasing pressure as demonstrated in the whole vessel STMs (B). Particle occurrence maps highlight the Ca²⁺ activity in each LMC as pressure is raised (C). Representative particle analysis plots for particle area (D) and particle counts/frame at each pressure (E). Summary files for particle area (F) and count /frame (G0. * Denotes p<0.05, Mean and SEM shown with n=12 separate IALVs from 8 MYH11-CreER^T2-GCaMP6f

Pressure-Dependent Diastolic Depolarization in LMCs
Intracellular recordings of LMC action potentials (AP) were confirmed by loading (greater than 10minutes) the impaling electrode with 1M KCl 100ug/ml AF488-Biocytin while recording APs followed by imaging on a spinning disk confocal microscope. 3D reconstruction of the z-stack confirmed the circumferential pattern of the impaled LMC that was strongly labeled by AF488-Biocytin (A, B), which also labeled neighboring LMCs, likely through gap junctions as AF488-Biocytin is <1kDa. In a separate set of experiments APs were recorded at 3 different pressures, 0.5 cmH₂O, 2 cmH₂O, and 5cmH₂O. We plotted the representative recordings from 1 cell at each pressure (C). AP frequency was significantly increased with pressure (D) as was the diastolic depolarization rate. Plotting the AP frequency and diastolic depolarization rate from all recordings at each pressure (F) highlights the significant effect diastolic depolarization rate has on the AP frequency. Minimum membrane potential (G), threshold membrane potential of AP initiation (H), upstroke constant (I), peak membrane potential (J), plateau membrane potential (K), and time over threshold (L) are also reported, although not significant.

Primer list for RT-PCR

Primer list for RT-PCR

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