Cellular characterization of the mouse collecting lymphatic vessels reveals that lymphatic muscle cells are the innate pacemaker cells
- Department of Medical Pharmacology & Physiology, University of Missouri, United States
- Bioinformatics and Analytics Core, Division of Research, Innovation and Impact, University of Missouri, United States
- Department of Pharmacology, Tulane University, United States
- Smooth Muscle Research Centre, Dundalk Institute of Technology, Ireland
Figures
Figure 1
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 (A). (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 to 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. Methylene blue staining was performed in IALVs isolated from five mice (n = 5).
Figure 2 with 3 supplements
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.
Figure 2—video 1
Expression of ICCLC markers in mouse IALVs.
Representative immunofluorescence imaging of a half vessel confocal image stack of IALV stained for CKIT (green), CD34 (red), and DAPI (blue) counterstained as shown in Figure 2D. Image acquisition at 1 µm step interval.
Figure 2—video 2
Expression of DESMIN in lymphatic muscle cells (LMCs) and CD34 in AdvCs.
Representative immunofluorescence imaging of a half vessel confocal image stack of IALV stained for DESMIN (green), CD34 (red), and VIMENTIN (cyan) as shown in Figure 2H. Image acquisition at 1 µm step interval.
Figure 2—video 3
CKIT and CD34 label separate cell populations in mouse IALVs.
Representative immunofluorescence imaging of a half vessel confocal stack of IALV stained for CKIT (green), CD34 (red), and VIMENTIN (cyan) as shown in Figure 2L. Image acquisition at 1 µm step interval.
Figure 3 with 4 supplements
Immunofluorescence labeling of mouse IALVs with markers for ICLC, lymphatic muscle cell (LMC), lymphatic endothelial cell (LEC), and immune cell populations.
We stained isolated mouse IALVs with cellular markers used to differentiate various cell types observed in collecting lymphatic vessels (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 ACTA2 (B) and CALPONIN (C) and the corresponding merged (D) image. There was significant overlap in (E) CD34 staining along with the fibroblast marker PDGFRα (F) compared to LMC staining with ACTA2 (G) and the merged (H) image. The endothelial marker PECAM1 (I) to delineate LECs with PDGFRα staining (J), and the LMC marker CALPONIN (K) with the merged image (L) revealed three 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 (R, S). Control IALV (T) stained only with secondary antibody. Scale bar = 100 µm for all images.
Figure 3—figure supplement 1
Colocalization of CD34 and PDGFRα.
Representative max projections and their corresponding threshold adjusted image for colocalization analysis for PDGFRα (A), CD34 (B), and their colocalized signal (C) and for comparison, we tested MYH11 (D) and PDGFRα (E) colocalization (F) using the FIJI BIOP-JACoP colocalization plugin on the z-stacks acquired by confocal microscopy. Pearson’s coefficient (G) and Mander’s coefficients (H) were calculated from n = 3 separate stained IALVS, each from a separate mouse for CD34 and PDGFRα and n = 4 for MYH11 and PDGFRα. Magnification for A–C ×40 and ×25 for D–F. Significant differences in colocalization below 0.05 are signified by the overhead lines.
Figure 3—figure supplement 2
PDGFRα+ cells reside primarily in the mouse lymphatic collecting vessel adventitia and some in the subendothelial space.
Max projection of confocal imaging of an IALV stained for lymphatic endothelial cells (LECs) with PECAM1 (A), lymphatic muscle cells (LMCs) with MYH11 (B), and for PDGFRα (C) with the corresponding merge file (D). Orthogonal views of the z-stack with (E) showing a single slice in the z stack and E’ and E” the orthogonal views. White dotted boxes outline locations where PDGFRα signal is observed between LMC and LEC layers. Scale bar is 100 µm in (D) and 50 µm in (E).
Figure 3—video 1
PDGFRα, CD34, and PDGFRβ label lymphatic valve interstitial cells.
Representative immunofluorescence confocal image stack of IALV stained for CD34 (green), PDGFRα (red), and PDGFRβ (blue) as shown in Figure 3R. Image acquisition at 1 µm step interval at lymphatic valve site.
Figure 3—video 2
Lymphatic valve interstitial cells have variable expression of PDGFRα, CD34, and PDGFRβ.
Representative immunofluorescence confocal image stack of IALV stained for CD34 (green), PDGFRα (red), and PDGFRβ (blue) as shown in Figure 3S. Image acquisition at 1 µm step interval at lymphatic valve site.
Figure 4
Inducible Cre-Rosa26mTmG labeling 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), Cspg4Cre-Rosa26mTmG (B), PdgfrαCreERTM-Rosa26mTmG (C), PdgfrβCreERT2-Rosa26mTmG (D), CkitCreERT2-Rosa26mTmG (E), and Myh11CreERT2-Rosa26mTmG (F). IALVs were digested into single cells and GFP+ cells were purified via FACS from Prox1-eGFP (G), Myh11CreERT2-Rosa26mTmG (H), PdgfrαCreERTM-Rosa26mTmG (I), and PdgfrβCreERT2-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 three separate mice (n = 3). FACS and RT-PCR were repeated at least three times (n = 3 mice).
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Figure 4—source data 1
This file contains the RT-PCR gel electrophoresis data for Figure 4.
- https://cdn.elifesciences.org/articles/90679/elife-90679-fig4-data1-v1.zip
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Figure 4—source data 2
This file contains the RT-PCR gel electrophoresis data for Figure 4 without markup.
- https://cdn.elifesciences.org/articles/90679/elife-90679-fig4-data2-v1.zip
Figure 5 with 5 supplements
scRNAseq analysis of mouse IALVs from Rosa26mTmG mice.
IALVs were cleaned and isolated from 10 Rosa26mTmG mice and digested into a single-cell suspension for scRNAseq analysis with the 10X platform. (A) Uniform manifold approximation and projection (UMAP) of the various cell populations that compromise the mouse IALV, though some mammary epithelia contamination was present (populations 18 and 19). (B) Heatmap of commonly used genes for cell identification for each of the cell clusters. Feature plots to assess cell cluster expression of the genes shown in Figure 4 inlcuding the lymphatic endothelial cell (LEC) markers Prox1 (C) and Flt4 (D), lymphatic muscle cell (LMC) markers Myh11 (E) and Calponin 1 (F, Cnn1), fibroblast markers Pdgfra (G) and Lum (H, Lumican), ICC marker Kit (I), the pericyte and smooth muscle precursor marker (Pdgfrb) (J), and the hematopoietic marker Ptprc (K).
Figure 5—figure supplement 1
scRNAseq analysis of the mouse IALV cell populations.
Heatmap of top 4–5 differentially expressed genes, based on p value, for each major cell cluster identified. Lymphatic endothelial cells (LECs) (clusters 0, 1, 2, 11), lymphatic muscle cells (LMCs) (clusters 5, 6), and IALV adventitial cells (AdvC, 3, 7, 8, 9, 10, 13) were comprised of multiple clusters. (B) Bubble plot of common identification genes reveals that the previously reported LMC transcriptome markers Dpt, Pi16, and Ackr3 are specific for a subpopulation of the AdvCs and not LMCs.
Figure 5—figure supplement 2
Subclusters of IALV lymphatic endothelial cells (LECs) revealed by scRNAseq.
The LECs were further subclustered to reveal 10 putative LEC subclusters (0–9) as shown in the uniform manifold approximation and projection (UMAP) (A) and the top differentially expressed genes amongst those subclusters are provided in the adjacent heatmap (B). (C) Bubble plot showing subcluster 8 was significantly enriched for previously documented LEC up-valve genes including Itga9, Cldn11, and Neo1 and cluster 6 had down-valve gene signature including Clu and Adm. The top 30 differentially expressed genes in cluster 8, both positive and negative fold change regulated, are labeled in the volcano plot(D).
Figure 5—figure supplement 3
Subclusters of IALV lymphatic muscle cells (LMCs) revealed by scRNAseq.
The LMCs could be subclustered into four putative subclusters (0–3) as shown in the uniform manifold approximation and projection (UMAP) (A). We profiled these subclusters based on their expression of the typical smooth muscle markers (B), SR-associated genes (C), voltage-gated Ca2+ channels (D), voltage-gated Na+ channels and Na+ transporters implicated in lymphatic pacemaking (E), voltage-gated K+ channels (F), Ca2+-activated K+ channels (G), inward-rectifying K+ channels and two-pore domain K+ channels (H), and Cl− channels (I).
Figure 5—figure supplement 4
Subclusters of IALV AdvCs revealed by scRNAseq.
AdvCs could also be further subclustered into multiple populations as shown in the uniform manifold approximation and projection (UMAP) (A). Bubble plot of genes used as Cre drivers and genes associated with pacemaking revealed subcluster 10 had expression of Ano1, Gjc1, and Cacna1c but with only minimal evidence of LMC contamination as indicated by muscle signature genes Myh11, Kcnma1, and Tagln (B). (C) Heatmap of the top differentially expressed genes among each of the subclusters. We assessed co-expression of Pdgfra with Cd34 (D) to confirm our immunofluorescence imaging (Figure 3—figure supplement 1) and assessed the co-expression of Pdgfra with the pericyte markers Pdgfrb (E) and Cspg4 (F). We further assessed co-expression of Pdgfra and the genes linked with contractile dysfunction Ano1 (G), Gcj1 (H), and Cacna1c (I) to ensure PdgfrαCreER would target the AdvCs expressing these genes. The cyan-colored slice of the pie chart indicates the minor population of cells expressing these genes that did not express Pdgfra.
Figure 5—figure supplement 5
Immune cell populations associated with the mouse IALV.
Lymphatic vessels are host to numerous immune cell populations, including monocyte, macrophage, and dendritic cell populations revealed by immunofluorescent staining for eGFP in the ‘Macgreen’ (Csf1r-eGFP) reporter mice (A). Staining for PDGFRα (B) demonstrates that AdvCs are distinct from the Csf1r-eGFP + cells, nor do they stain for the hematopoietic marker PTPRC (also known as CD45) (C, D). Bubble plot of our scRNASeq analysis of IALVs revealed macrophages (cluster 4), moDCs and cDC2 (cluster 14), and cDC1 cells (cluster 17) based off identifying gene markers (B). (C) Bubble plot of T-cell markers revealed multiple populations of T cells including naive double-negative T-cells (Yang et al., 2021) and naive Cd4+ and Cd8+ T-cells. A bubble plot for B-cell markers showed that cluster 15 had an expression profile for immature and mature B2 B-cells (D) (Luo et al., 2022a).
Figure 6 with 2 supplements
RT-PCR Profiling of FACS-purified cells from inducible Cre-Rosa26mTmG.
Expanded RT-PCR profiling of genes to discriminate lymphatic endothelial cells (LECs), lymphatic muscle cells (LMCs), and other cell types in our GFP+ sorted cells from Prox1-eGFP (A), Myh11CreERT2-Rosa26mTmG (B), PdgfrβCreERT2-Rosa26mTmG (C), and PdgfrαCreERTM-Rosa26mTmG (D). Feature plots for the genes assessed in A-D in our IALV scRNAseq analysis confirmed those results (E-M). In addition to a population of AdvCs expressing Cacna1c, we also noted expression of Gjc1 (N), which was also observed in LECs, and Ano1 (O) in the AdvC clusters. We confirmed this expression using GFP+ cells sorted from PdgfrαCreERTM-Rosa26mTmG IALVs for RT-PCR (P) and ruled out hematopoietic or LEC contamination. All RT-PCRs were performed two to four times for each gene over each sorted cell population collected from different mice.
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Figure 6—source data 1
This file contains the RT-PCR gel electrophoresis data for Figure 6 without markup.
- https://cdn.elifesciences.org/articles/90679/elife-90679-fig6-data1-v1.zip
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Figure 6—source data 2
This file contains the RT-PCR gel electrophoresis data for Figure 6 without markup.
- https://cdn.elifesciences.org/articles/90679/elife-90679-fig6-data2-v1.zip
Figure 6—figure supplement 1
PDGFRα AdvCs include multipotent cell.
Representative RT-PCR results profiling purified GFP+ cells purified from IALVs isolated from PdgfrαCreERTM-Rosa26mTmG via FACS. PDGFRα cells expressed the multipotent markers Klf4, Ly6a, Gli1, Itgb1, Eng, and Cd44 (A) with total brain cDNA serving as a positive control (B). Representative RT-PCR results showing lack of expression of some of these markers in the GFP+ cells purified from Myh11CreERT2-Rosa26mTmG (C) or Prox1-eGFP mice, in contrast to the RFP+ population from Myh11CreERT2-Rosa26mTmG mice (D). RT-PCRs were repeated at least two times from separate purified cell populations from different mice. Feature plots of only the AdvCs cluster highlight populations of cells that express genes associated with multipotency such as Ly6a (E), Klf4 (F), Gli1 (G), Itgb1 (H), Eng (I), Cd44 (J). Expression of LY6A protein was confirmed with immunofluorescence. Representative max projections of IALVs stained for LY6A (K), PDGFRα (L), MYH11 (M), and the corresponding merged file (N). Scale bar is 100 µm.
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Figure 6—figure supplement 1—source data 1
This file contains the RT-PCR gel electrophoresis data for Figure 6—figure supplement 1.
- https://cdn.elifesciences.org/articles/90679/elife-90679-fig6-figsupp1-data1-v1.zip
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Figure 6—figure supplement 1—source data 2
This file contains the RT-PCR gel electrophoresis data for Figure 6—figure supplement 1 without markup.
- https://cdn.elifesciences.org/articles/90679/elife-90679-fig6-figsupp1-data2-v1.zip
Figure 6—video 1
Lymphatic PDGFRα+ AdvCs co-stain with the stem cell marker LY6A.
Representative immunofluorescence imaging of a half vessel confocal image stack of IALV stained for LY6A (green), PDGFRα (red), and MYH11 (blue) as shown in Figure 6—figure supplement 1N. Image acquisition at 1 µm step interval.
Figure 7 with 1 supplement
Isobaric contractile assessment of popliteal collecting lymphatic vessel (cLV) from PdgfrαCreERTM-driven deletion of Ano1, GJC1, and Cacna1c.
Summary of the contractile parameters recorded from popliteal cLVs in PdgfrαCreERTM-Ano1fl/fl, PdgfrαCreERTM-Gjc1fl/fl mice, PdgfrαCreERTM-Cacna1cfl/fl mice. Contraction frequency (A, D, G), ejection fraction (B, E, H), and vessel tone (C, F, I) were assessed. No statistically significant differences were observed in cLVs isolated from PdgfrαCreERTM-Ano1fl/fl and PdgfrαCreERTM-Gjc1fl/fl mice across these three parameters. Mean and SEM shown, n = 6 popliteal vessels from three mice PdgfrαCreERTM-Ano1fl/fl mice and n = 9 popliteal vessels from six mice Ano1fl/fl mice. Mean and SEM shown, n = 5 popliteal vessels from three mice PdgfrαCreERTM-GJC1fl/fl mice and n = 11 popliteal vessels from eight mice GJC1fl/fl mice. Mean and SEM shown, n = 6 popliteal vessels from three mice PdgfrαCreERTM-Cacna1cfl/fl mice and n = 8 popliteal vessels from eight mice Cacna1cfl/fl mice. The contractile data from control Cacna1cfl/fl vessels is a subset of previously published data that 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 Figure 7—figure supplement 1.
Figure 7—figure supplement 1
Contractile indices from isobaric myography on collecting lymphatic vessels (cLVs) from PdgfrαCreERTM-driven deletion of Ano1, GJC1, and Cacna1c.
Summary of the contractile parameters recorded from popliteal cLVs in PdgfrαCreERTM-Ano1fl/fl, PdgfrαCreERTM-Gjc1fl/fl mice, PdgfrαCreERTM-Cacna1cfl/fl mice. No differences in normalized contraction amplitude (A, D, G), fractional pump flow (B, E, H), or end diastolic diameter (C, F, J) were observed. The contractile data from control Cacna1cfl/fl vessels is a subset of previously published data that was separated by sex (Davis et al., 2022) while they are combined here. Mean and SEM shown, n = 6 popliteal vessels from three mice PdgfrαCreERTM-Ano1fl/fl mice and n = 9 popliteal vessels from six mice Ano1fl/fl mice. Mean and SEM shown, n = 5 popliteal vessels from three mice PdgfrαCreERTM-GJC1fl/fl mice and n = 11 popliteal vessels from eight mice Gjc1fl/fl mice. Mean and SEM shown, n = 6 popliteal vessels from three mice PdgfrαCreERTM-Cacna1cfl/fl mice and n = 8 popliteal vessels from eight mice Cacna1cfl/fl mice.
Figure 8 with 3 supplements
ChR2-mediated depolarization only in lymphatic muscle cells (LMCs) triggers contraction.
Representative max projections of tdTomato-ChR2 signal in popliteal collecting lymphatic vessels (cLVs) isolated from CkitCreERT2-ChR2-tdTomato (A), PdgfrαCreERTM-ChR2-tdTomato (C), and Myh11CreERT2-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 CkitCreERT2-ChR2-tdTomato (G–J), PdgfrαCreERTM-ChR2-tdTomato (K–N), and Myh11CreERT2-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 demonstrates spontaneous contractions with the dotted boxes indicating the optical stimulation event in the respective brightfield images of the time lapse images. Isolated cLVs from CkitCreERT2-ChR2-tdTomato (S), PdgfrαCreERTM-ChR2-tdTomato (T), and Myh11CreERT2-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 six popliteal cLVs from 3 mice per genotype.
Figure 8—video 1
Brightfield video of optogenetic stimulation of IALVs from CkitCreERT2-ChR2 mice.
Isolated, cannulated, and spontaneously contracting popliteal lymphatic collecting lymphatic vessel (cLV) from a CkitCreERT2-ChR2 mouse was stimulated with light at regions of ChR2 expression, as confirmed by tdTomato imaging, but these stimulations failed to initiate a contraction. Vessels maintained spontaneous contractile activity, but contraction frequency was depressed with application of 100 nM pinacidil to ensure sufficient time in diastole for photo-stimulation. Video of the representative trace in Figure 8.
Figure 8—video 2
Brightfield video of optogenetic stimulation of IALVs from PdgfrαCreER-ChR2 mice.
Isolated, cannulated, and spontaneously contracting popliteal lymphatic collecting lymphatic vessel (cLV) from a PdgfrαCreER-ChR2 mouse was stimulated with light at regions of ChR2 expression, as confirmed by tdTomato imaging, but these stimulations failed to initiate a contraction. Vessels maintained spontaneous contractile activity, but contraction frequency was depressed by the application of 100 nM pinacidil to ensure sufficient time in diastole for photo-stimulation. Video of the representative trace in Figure 8.
Figure 8—video 3
Brightfield video of optogenetic stimulation of IALVs from Myh11CreERT2-ChR2 mice.
Isolated, cannulated, and spontaneously contracting popliteal lymphatic collecting lymphatic vessel (cLV) isolated from a Myh11CreERT2-ChR2 mouse was stimulated with light in an attempt to drive a propagated contraction from the target cell. Photo-stimulation of tdTomato+ lymphatic muscle cells (LMCs) initiated a coordinated and propagated contraction similar to a spontaneous contraction. Vessels maintained spontaneous contractile activity, but contraction frequency was depressed by the application of 100 nM pinacidil to ensure sufficient time in diastole for photo-stimulation. Video of the representative trace in Figure 8.
Figure 9 with 1 supplement
CkitCreERT2 drives GCaMP6f expression primarily in mast cells in mouse IALVs.
Representative max projection (A) of GCaMP6f signal over time in an IALV isolated from a CkitCreERT2-GCaMP6f mouse with ROI indicated around individual cells, primarily large ovoid cells, but also including a circumferential lymphatic muscle cell (LMC) (cell 10) and a horizontal lymphatic endothelial cell (LEC) (cell 11). Of cells 1–9, only cell 7 had any Ca2+ activity (red arrows) during the recording time as indicated by the spatio-temporal maps (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). Calcium recordings were made in n = 6 IALVs from four mice.
Figure 9—video 1
Calcium imaging in an IALV isolated from a CkitCreERT2-GCaMP6f mouse.
GCaMP6f signal was readily detected in ovoid cells and occasionally in circumferential lymphatic muscle cells (LMCs) and longitudinal lymphatic endothelial cells (LECs). The ovoid cells did not have consistent calcium transients, while circumferential LMCs displayed global calcium flashes in a near synchronous manner. Vessel contractions were mitigated with 2 µM wortmannin to maintain the vessel wall in the focal plane.
Figure 10 with 1 supplement
Lack of coordinated Ca2+ activity across contraction cycle in PDGFRα cells.
Representative max projections of GCaMP6f signal over time in IALVs isolated from PdgfrαCreERTM-GCaMP6f mice (A, D). ROIs were made around cells and GCaMP6f recorded over time to generate the corresponding spatio-temporal maps (STMs) (B, E) for each cell and plots (C, F), respectively. Once again, incidental recombination occurred in a lymphatic muscle cell (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 PdgfrαCreERTM-GCaMP6f IALV. Calcium recordings were made in n = 6 IALVs from four mice.
Figure 10—video 1
Calcium imaging in an IALV isolated from a PdgfrαCreER-GCaMP6f mouse.
Calcium imaging in an IALV isolated from a PdgfrαCreER-ChR2 mouse. GCaMP6f signal was readily detected in the AdvCs along the length of the IALV. Very few calcium transients were observed in these cells across the vessel contraction cycle. In contrast, lymphatic muscle cells (LMCs) with incidental recombination displayed the classic rhythmic calcium flash that precedes each contraction. Vessel contractions were mitigated with 2 µM wortmannin to maintain the vessel wall in the focal plane.
Figure 11 with 1 supplement
Heterogeneous diastolic Ca2+ transient activity in lymphatic muscle cells (LMCs).
Representative max projections of GCaMP6f signal over time in an IALVs isolated from Myh11CreERT2-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). Representative images from calcium recordings from n = 4 IALVs from four mice.
Figure 11—video 1
Calcium imaging in an IALV isolated from a Myh11CreERT2-GCaMP6f mouse.
Calcium imaging in an IALV isolated from a Myh11CreERT2-ChR2 mouse. CaMP6f signal was readily detected in circumferential lymphatic muscle cells (LMCs) along the length of the IALV. Each LMC participated in nearly synchronous calcium flash that propagated down the length of the vessel prior to each contraction. There was heterogeneity in the prevalence of subcellular calcium transients in diastole. Vessel contractions were mitigated with 2 µM wortmannin to maintain the vessel wall in the focal plane.
Figure 12 with 3 supplements
Pressure dependency of mouse lymphatic muscle cell (LMC) diastolic Ca2+ transients.
Representative max projection of GCaMP6f signal over 20 s in an IALVs isolated from Myh11CreERT2-GCaMP6f mice in the presence of the L-type blocker nifedipine (1 μM) (A) and pressurized to 0.5, 2, and 5 cmH2O. The local diastolic Ca2+ transients persist in the presence of nifedipine and increase with increasing pressure as demonstrated in the whole vessel spatio-temporal maps (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 (G). * denotes p < 0.05, Mean and SEM shown with n = 12 separate IALVs from 8 Myh11CreERT2-GCaMP6f.
Figure 12—video 1
Imaging subcellular calcium oscillations, at a pressure of 0.5 cmH2O, in an IALV isolated from a Myh11CreERT2-GCaMP6f mouse.
Subcellular calcium oscillations in lymphatic muscle cells (LMCs) persist in the presence of 1 µM nifedipine. The activity is variable across neighboring LMCs. In contrast to the nearly synchronous global calcium flash events, the subcellular calcium transients are not conducted across cells.
Figure 12—video 2
Imaging subcellular calcium oscillations, at a pressure of 2.0 cmH2O, in an IALV isolated from a Myh11CreERT2-GCaMP6f mouse.
Subcellular calcium oscillations in lymphatic muscle cells (LMCs) persist in the presence of 1 µM nifedipine. The activity is variable across neighboring LMCs and is increased as compared to 0.5 cmH2O. In contrast to the nearly synchronous global calcium flash events, the subcellular calcium transients are not conducted across cells.
Figure 12—video 3
Imaging subcellular calcium oscillations, at a pressure of 5.0 cmH2O, in an IALV isolated from a Myh11CreERT2-GCaMP6f mouse.
Subcellular calcium oscillations in lymphatic muscle cells (LMCs) persist in the presence of 1 µM nifedipine. The activity is variable across neighboring LMCs, but now the majority of LMCs are displaying calcium transient oscillations with an activity that is further increased as compared to 2.0 cmH2O. In contrast to the nearly synchronous global calcium flash events, the subcellular calcium transients are not conducted across cells.
Figure 13
Pressure-dependent diastolic depolarization in lymphatic muscle cells (LMCs).
Intracellular recordings of LMC action potentials (APs) were confirmed by loading (greater than 10 min) the impaling electrode with 1 M KCl 100 µg/ml AF488-Biocytin while recording APs followed by imaging on a spinning disk confocal microscope (n = 3 vessels from 3 mice). 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 <1 kDa. In a separate set of experiments APs were recorded at three different pressures, 0.5, 2, and 5 cmH2O. We plotted the representative recordings from one cell at each pressure (C). AP frequency was significantly increased with pressure (D) as was the diastolic depolarization rate (E). 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. Recordings are from 10 IALVs from 10 mice.
Tables
Table 1
Primer list for RT-PCR.
| Gene | Strand | Accession # | Sequence (5'–3') | Size | Exon | Source |
|---|---|---|---|---|---|---|
| Prox1 | s | NM_008937 | GTA AGA CAT CAC CGC GTG C | 218 | 1 | NIH Primer Tool |
| as | TCA TGG TCA GGC ATC ACT GG | 2 | ||||
| Itgam (Cd11b) | s | NM_008401 | ATG GAC GCT GAT GGC AAT ACC | 203 | 13 | MGH Primer Bank ID 668048a1 |
| as | TCC CCA TTC ACG TCT CCC A | 14 | ||||
| Pdgfra | s | NM_011058 | AGA GTT ACA CGT TTG AGC TGT C | 252 | 8 | MGH Primer Bank 26349287a1 |
| as | GTC CCT CCA CGG TAC TCC T | 10 | ||||
| Myh11 | s | NM_013607 | AAG CTG CGG CTA GAG GTC A | 238 | 33 | MGH Primer Bank ID 7305295a1 |
| as | CCC TCC CTT TGA TGG CTG AG | 34 | ||||
| cKit (Cd117) | s | NM_021099 | CGC CTG CCG AAA TGT ATG ACG | 162 | 21 | Drumm et al., 2018 |
| as | GGT TCT CTG GGT TGG GGT TGC | 23 | ||||
| Pdgfrb | s | NM_008809 | AGC TAC ATG GCC CCT TAT GA | 367 | 16 | Basciani et al., 2004 |
| as | GGA TCC CAA AAG ACC AGA CA | 19 | ||||
| Cdh5 (Cadherin, VE-cadherin) | s | NM_009868 | CTT CCT TAC TGC CCT CAT TGT | 313 | 3 | IDT Primer Quest |
| as | CTG TTT CTC TCG GTC CAA GTT | 5 | ||||
| Nos3 (eNOS) | s | NM_008713 | CTG CCA CCT GAT CCT AAC TTG | 143 | 22 | IDT Real time primer tool |
| as | CAG CCA AAC ACC AAA GTC ATG | 23 | ||||
| Acta2 (Smooth Muscle Actin) | s | NM_007392 | GAG CTA CGA ACT GCC TGA C | 129 | 7 | IDT TaqMan Mm.PT.58.16320644 |
| as | CTG TTA TAG GTG GTT TCG TGG A | 8 | ||||
| Cacna1c exon1b | s | NM_001159533 | ATG GTC AAT GAA AAC ACG AGG ATG | 1 | Cheng et al., 2007 | |
| as | GGA ACT GAC GGT AGA GAT GGT TGC | 234 | 2 | |||
| Cd34 | as | NM_133654 | GGT ACA GGA GAA TGC AGG TC | 119 | 1 | IDT Mm.PT.58.8626728 |
| s | CGT GGT AGC AGA AGT CAA GT | 2 | ||||
| Cspg4 (Ng2) | as | NM_139001 | CTT CAC GAT CAC CAT CCT TCC | 132 | 5 | IDT Mm.PT.58.29461721 |
| s | CCC GAA TCA TTG TCT GTT CCC | 6 | ||||
| Vimentin | s | NM_011701 | CTG TAC GAG GAG GAG ATG CG | 249 | 1 | Li et al., 2016 |
| as | AAT TTC TTC CTG CAA GGA TT | 3 | ||||
| Desmin | s | NM_010043 | GTG GAT GCA GCC ACT CTA GC | 218 | 3 | MGH Primer Bank ID 33563250a1 |
| as | TTA GCC GCG ATG GTC TCA TAC | 4 | ||||
| Mcam (Cd146) | s | NM_023061 | CCC AAA CTG GTG TGC GTC TT | 220 | 1 | MGH Primer Bank 10566955a1 |
| as | GGA AAA TCA GTA TCT GCC TCT CC | 3 | ||||
| Klf4 | s | NM_010637 | ATT AAT GAG GCA GCC ACC TG | 400 | 1 | Majesky et al., 2017 |
| as | GGA AGA CGA GGA TGA AGC TG | 3 | ||||
| Ly6a (Sca1) | s | NM_001271416 | CTC TGA GGA TGG ACA CTT CT | 400 | 2 | Majesky et al., 2017 |
| as | GGT CTG CAG GAG GAC TGA GC | 4 | ||||
| Gli1 | s | NM_01029 | ATC ACC TGT TGG GGA TGC TGG AT | 316 | 8 | Kramann et al., 2015 |
| as | CGT GAA TAG GAC TTC CGA CAG | 10 | ||||
| Itgb1 (Cd29) | s | NM_010578 | TCG ATC CTG TGA CCC ATT GC | 170 | 14 | NIH Primer Tool |
| as | AAC AAT TCC AGC AAC CAC GC | 15 | ||||
| Endoglin (Eng, Cd105) | s | NM_007932 | TGA GCG TGT CTC CAT TGA CC | 416 | 11 | NIH Primer Tool |
| as | GGG GCC ACG TGT GTG AGA A | 15 | ||||
| Cd44 | s | NM_009851 | CAC CAT TTC CTG AGA CTT GCT | 148 | 18 | IDT Mm.PT.58.12084136 |
| as | TCT GAT TCT TGC CGT CTG C | 19 | ||||
| Pecam1 (Cd31) | s | NM_008816 | CTG CCA GTC CGA AAA TGG AAC | 218 | 7 | MGH Primer Bank ID 6679273a1 |
| as | CTT CAT CCA CTG GGG CTA TC | 8 | ||||
| Gjc1 (Connexin 45) | s | NM_008122 | GGT AAC AGG AGT TCT GGT GAA | 140 | 2 | IDT Mm.PT.58.8383900 |
| as | TCG AAA GAC AAT CAG CAC AGT | 3 | ||||
| Anoctamin 1 (TMEM16A) | s | NM_178642 | GGC ATT TGT CAT TGT CTT CCA G | 141 | 25 | IDT Real time primer tool |
| as | TCC TCA CGC ATA AAC AGC TC | 26 | ||||
| Ptprc (Cd45) | s | NM_001111316 | ATG CAT CCA TCC TCG TCC AC | 225 | 29 | NIH Primer Tool |
| as | TGA CTT GTC CAT TCT GGG CG | 31 | ||||
| MGH Harvard Primer Bank (Wang and Seed, 2003; Spandidos et al., 2008; Spandidos et al., 2010). | ||||||
Appendix 1—key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background (Mus musculus) | C57BL/6J | The Jackson Laboratory | Jax Strain #000664 RRID:IMSR_JAX:000664 | |
| Strain, strain background (Mus musculus) | Rosa26mTmG | The Jackson Laboratory | Jax Strain #007676 B6.129(Cg)-Gt(ROSA)26Sortm4(ACTB-tdTomato,-EGFP) Luo/J RRID:IMSR_JAX:007676 | |
| Strain, strain background (Mus musculus) | PdgfrαCre | The Jackson Laboratory | Jax Strain #013148 C57BL/6-Tg(Pdgfra-cre)1Clc/J RRID:IMSR_JAX: 013148 | |
| Strain, strain background (Mus musculus) | Csfr1-EGFP | The Jackson Laboratory | Jax Strain #018549 B6.Cg-Tg(Csf1r-EGFP)1Hume/J RRID:IMSR_JAX:018549 | |
| Strain, strain background (Mus musculus) | PdgfrαCreERTM | The Jackson Laboratory | Jax Strain #018280 B6N.Cg-Tg(Pdgfra-cre/ERT)467Dbe/J RRID:IMSR_JAX:018280 | |
| Strain, strain background (Mus musculus) | Cspg4-Cre | The Jackson Laboratory | Jax Strain #:008533 B6;FVB-Ifi208Tg(Cspg4-cre)1Akik/J RRID:IMSR_JAX:008533 | |
| Strain, strain background (Mus musculus) | ChR2/tdTomato | The Jackson Laboratory | Jax Strain #012567 B6.Cg-Gt(ROSA)26Sortm27.1(CAG-COP4*H134R/tdTomato)Hze/J RRID:IMSR_JAX:012567 | |
| Strain, strain background (Mus musculus) | PdgfrβCreERT2 | The Jackson Laboratory | Jax Strain #029684 B6.Cg-Tg(Pdgfrb-cre/ERT2)6096Rha/J RRID:IMSR_JAX:029684 | |
| Strain, strain background (Mus musculus) | Myh11CreERT2 | The Jackson Laboratory | Jax Strain #019079 B6.FVB-Tg(Myh11-icre/ERT2)1Soff/J RRID:IMSR_JAX:019079 | |
| Strain, strain background (Mus musculus) | CkitCreERT2 | Dieter Saur (Technical University of Munich) | Kittm1(cre/ERT2)Dsa | |
| Strain, strain background (Mus musculus) | Prox1-eGFP | Young-Kwon Hong (University of Southern California) | MMRRC ID #31006 Tg(Prox1-EGFP)KY221Gsat/Mmucd RRID:MMRRC_031006-UCD | |
| Strain, strain background (Mus musculus) | GCaMP6f | Jax | Jax Strain #028865 Ai95(RCL-GCaMP6f)-D (C57BL/6J) or Ai95D (C57BL/6J) RRID:IMSR_JAX:028865 | |
| Antibody | anti-ACTA2 | Sigma-Aldrich | Cat# A2547, RRID:AB_476701 | (IF) 1:500 |
| Antibody | anti-GFP | Thermo Fisher Scientific | Cat# A-11122, RRID:AB_221569 | (IF) 1:200 |
| Antibody | anti-CKIT | Cell Signaling | Cell Signaling Technology Cat# 3074, RRID:AB_1147633 | (IF) 1:100 |
| Antibody | anti-VIMENTIN | Thermo Fisher Scientific | Cat# OMA1-06001, RRID:AB_325529 | (IF) 1:100 |
| Antibody | anti-DESMIN | Invitrogen | Cat# PA5-16705, RRID:AB_10977258 | (IF) 1:200 |
| Antibody | anti-GFP | Abcam | Cat# ab13970, RRID:AB_300798 | (IF) 1:200 |
| Antibody | anti-CD34 (RAM34) | Thermo Fisher Scientific | Cat# 14-0341-82, RRID:AB_467210 | (IF) 1:200 |
| Antibody | anti-PDGFRα | R&D Systems | Cat# AF1062, RRID:AB_2236897 | (IF) 1:200 |
| Antibody | anti-PDGFRβ | eBiosciences | Cat# 14-1402-82, RRID:AB_467493 | (IF) 1:200 |
| Antibody | anti-CALPONIN | Abcam | Cat# ab46794, RRID:AB_2291941 | (IF) 1:500 |
| Antibody | anti-LY6A | Biolegend | Cat# 108101, RRID:AB_313338 | (IF) 1:200 |
| Sequence-based reagent | Prox1- Forward NM_008937 | NIH Primer Tool, this paper | PCR primers | GTA AGA CAT CAC CGC GTG C |
| Sequence-based reagent | Prox1- Reverse NM_008937 | NIH Primer Tool, this paper | PCR primers | TCA TGG TCA GGC ATC ACT GG |
| Sequence-based reagent | Itgam Reverse NM_008401 | MGH Primer Bank ID 668048a1 | PCR primers | ATG GAC GCT GAT GGC AAT ACC |
| Sequence-based reagent | Itgam Forward NM_008401 | MGH Primer Bank ID 668048a1 | PCR primers | TCC CCA TTC ACG TCT CCC A |
| Sequence-based reagent | Pdgfra Forward NM_011058 | MGH Primer Bank ID 26349287a1 | PCR primers | AGA GTT ACA CGT TTG AGC TGT C |
| Sequence-based reagent | Pdgfra Reverse NM_011058 | MGH Primer Bank ID 26349287a1 | PCR primers | GTC CCT CCA CGG TAC TCC T |
| Sequence-based reagent | Myh11 Forward NM_013607 | MGH Primer Bank ID 7305295a1 | PCR primers | AAG CTG CGG CTA GAG GTC A |
| Sequence-based reagent | Myh11 Reverse NM_013607 | MGH Primer Bank ID 7305295a1 | PCR primers | CCC TCC CTT TGA TGG CTG AG |
| Sequence-based reagent | Ckit Forward NM_021099 | Drumm et al., 2018 | PCR primers | CGC CTG CCG AAA TGT ATG ACG |
| Sequence-based reagent | Ckit Reverse NM_021099 | Drumm et al., 2018 | PCR primers | GGT TCT CTG GGT TGG GGT TGC |
| Sequence-based reagent | Pdgfrb Forward NM_008809 | Basciani et al., 2004 | PCR primers | AGC TAC ATG GCC CCT TAT GA |
| Sequence-based reagent | Pdgfrb Reverse NM_008809 | Basciani et al., 2004 | PCR primers | GGA TCC CAA AAG ACC AGA CA |
| Sequence-based reagent | Cdh5 Forward NM_009868 | IDT Primer Quest Tool, this paper | PCR primers | CTT CCT TAC TGC CCT CAT TGT |
| Sequence-based reagent | Cdh5 Reverse NM_009868 | IDT Real time primer tool, this paper | PCR primers | CTG TTT CTC TCG GTC CAA GTT |
| Sequence-based reagent | Nos3 Forward NM_008713 | IDT Real time primer tool, this paper | PCR primers | CTG CCA CCT GAT CCT AAC TTG |
| Sequence-based reagent | Nos3 Reverse NM_008713 | IDT Real time primer tool, this paper | PCR primers | CAG CCA AAC ACC AAA GTC ATG |
| Sequence-based reagent | Acta2 (Smooth Muscle Actin) Forward NM_007392 | IDT TaqMan Mm.PT.58.16320644 | PCR primers | GAG CTA CGA ACT GCC TGA C |
| Sequence-based reagent | Acta2 (Smooth Muscle Actin) Reverse NM_007392 | IDT TaqMan Mm.PT.58.16320644 | PCR primers | CTG TTA TAG GTG GTT TCG TGG A |
| Sequence-based reagent | Cacna1c (CaV 1.2) Forward NM_001159533 | Cheng et al., 2007 | PCR primers | ATG GTC AAT GAA AAC ACG AGG ATG |
| Sequence-based reagent | Cacna1c (CaV 1.2) Reverse NM_001159533 | Cheng et al., 2007 | PCR primers | GGA ACT GAC GGT AGA GAT GGT TGC |
| Sequence-based reagent | Cd34 Forward NM_133654 | IDT Mm.PT.58.8626728 | PCR primers | GGT ACA GGA GAA TGC AGG TC |
| Sequence-based reagent | Cd34 Reverse NM_133654 | IDT Mm.PT.58.8626728 | PCR primers | CGT GGT AGC AGA AGT CAA GT |
| Sequence-based reagent | Cspg4 (Ng2) Forward NM_139001 | IDT Mm.PT.58.29461721 | PCR primers | CTT CAC GAT CAC CAT CCT TCC |
| Sequence-based reagent | Cspg4 (Ng2) Reverse NM_139001 | IDT Mm.PT.58.29461721 | PCR primers | CCC GAA TCA TTG TCT GTT CCC |
| Sequence-based reagent | Vimentin Forward NM_011701 | Li et al., 2016 | PCR primers | CTG TAC GAG GAG GAG ATG CG |
| sequence-based reagent | Vimentin Reverse NM_011701 | Li et al., 2016 | PCR primers | AAT TTC TTC CTG CAA GGA TT |
| Sequence-based reagent | Desmin Forward NM_010043 | MGH Primer Bank ID 33563250a1 | PCR primers | GTG GAT GCA GCC ACT CTA GC |
| Sequence-based reagent | Desmin Reverse NM_010043 | MGH Primer Bank ID 33563250a1 | PCR primers | TTA GCC GCG ATG GTC TCA TAC |
| Sequence-based reagent | Mcam Forward NM_023061 | MGH Primer Bank ID 10566955a1 | PCR primers | CCC AAA CTG GTG TGC GTC TT |
| Sequence-based reagent | Mcam Reverse NM_023061 | MGH Primer Bank ID 10566955a1 | PCR primers | GGA AAA TCA GTA TCT GCC TCT CC |
| Sequence-based reagent | Klf4 Forward NM_010637 | Majesky et al., 2017 | PCR primers | ATT AAT GAG GCA GCC ACC TG |
| Sequence-based reagent | Klf4 Reverse NM_010637 | Majesky et al., 2017 | PCR primers | GGA AGA CGA GGA TGA AGC TG |
| Sequence-based reagent | Ly6a Forward NM_001271416 | Majesky et al., 2017 | PCR primers | CTC TGA GGA TGG ACA CTT CT |
| Sequence-based reagent | Ly6a Reverse NM_001271416 | Majesky et al., 2017 | PCR primers | GGT CTG CAG GAG GAC TGA GC |
| Sequence-based reagent | Gli1 Forward NM_01029 | Kramann et al., 2015 | PCR primers | ATC ACC TGT TGG GGA TGC TGG AT |
| Sequence-based reagent | Gli1 Reverse NM_01029 | Kramann et al., 2015 | PCR primers | CGT GAA TAG GAC TTC CGA CAG |
| Sequence-based reagent | Itgb1 Forward NM_010578 | NIH Primer Tool, this paper | PCR primers | TCG ATC CTG TGA CCC ATT GC |
| Sequence-based reagent | Itgb1 Reverse NM_010578 | NIH Primer Tool, this paper | PCR primers | AAC AAT TCC AGC AAC CAC GC |
| Sequence-based reagent | Eng Forward NM_007932 | NIH Primer Tool, this paper | PCR primers | TGA GCG TGT CTC CAT TGA CC |
| Sequence-based reagent | Eng Reverse NM_007932 | NIH Primer Tool, this paper | PCR primers | GGG GCC ACG TGT GTG AGA A |
| Sequence-based reagent | Cd44 Forward NM_009851 | IDT Mm.PT.58.12084136 | PCR primers | CAC CAT TTC CTG AGA CTT GCT |
| Sequence-based reagent | Cd44 Reverse NM_009851 | IDT Mm.PT.58.12084136 | PCR primers | TCT GAT TCT TGC CGT CTG C |
| Sequence-based reagent | Pecam1 Forward NM_008816 | MGH Primer Bank ID 6679273a1 | PCR primers | CTG CCA GTC CGA AAA TGG AAC |
| Sequence-based reagent | Pecam1 Reverse NM_008816 | MGH Primer Bank ID 6679273a1 | PCR primers | CTT CAT CCA CTG GGG CTA TC |
| Sequence-based reagent | Gjc1 Forward NM_008122 | IDT Mm.PT.58.8383900 | PCR primers | GGT AAC AGG AGT TCT GGT GAA |
| Sequence-based reagent | Gjc1 Reverse NM_008122 | IDT Mm.PT.58.8383900 | PCR primers | TCG AAA GAC AAT CAG CAC AGT |
| Sequence-based reagent | Anoctamin 1 Forward NM_178642 | IDT Real time primer tool, this paper | PCR primers | GGC ATT TGT CAT TGT CTT CCA G |
| Sequence-based reagent | Anoctamin 1 Reverse NM_178642 | IDT Real time primer tool, this paper | PCR primers | TCC TCA CGC ATA AAC AGC TC |
| Sequence-based reagent | Ptprc Forward NM_001111316 | NIH Primer Tool, this paper | PCR primers | ATG CAT CCA TCC TCG TCC AC |
| Sequence-based reagent | Ptprc Reverse NM_001111316 | NIH Primer Tool, this paper | PCR primers | TGA CTT GTC CAT TCT GGG CG |
| Chemical compound, drug | Methylene Blue | Sigma | M9140 | |
| Chemical compound, drug | Blockaid | Thermo Fisher | A11122 | |
| Peptide, recombinant protein | Collagenase H | Sigma | C8051 | |
| Peptide, recombinant protein | Collagenase F | Sigma | C7926 | |
| Chemical compound, drug | Dithioerythritol (DTT) | Sigma | D8161 | |
| Peptide, recombinant protein | Elastase | Worthington | LS00635 | |
| Peptide, recombinant protein | Papain | Sigma | P4762 | |
| Antibody | Donkey anti-mouse AF647 | Thermo Fisher | A32787 | (IF) 1:200 |
| Antibody | Donkey anti-Rat AF555 | Thermo Fisher | A48270 | (IF) 1:200 |
| Antibody | Donkey anti-Rabbit AF488 | Thermo Fisher | A21206 | (IF) 1:200 |
| Antibody | Donkey anti-Goat AF647 | Thermo Fisher | A21447 | (IF) 1:200 |
| Antibody | Donkey anti-Goat AF555 | Thermo Fisher | A21432 | (IF) 1:200 |
| Commercial assay or kit | Arcturus PicoPure RNA isolation kit | Thermo Fisher | KIT0204 | |
| Software, algorithm | Prism 10 | GraphPad | ||
| Software, algorithm | Volumetry software (version G8d) | Grant Hennig | Drumm et al., 2017; Drumm et al., 2019a | |
| Software, algorithm | Seurat v4,v5 | RRID:SCR_016341 | Hao et al., 2021; Hao et al., 2024 | |
| Software, algorithm | FIJI BIOP-JACoP | Bolte and Cordelières, 2006 |
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Cellular characterization of the mouse collecting lymphatic vessels reveals that lymphatic muscle cells are the innate pacemaker cells
eLife 12:RP90679.
https://doi.org/10.7554/eLife.90679.4
