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

Collecting lymphatic vessels (cLVs) exhibit spontaneous contractions with a pressure-dependent frequency, but the identity of the lymphatic pacemaker cell is still debated. By analogy to pacemakers in the GI and lower urinary tracts, proposed cLV pacemaker cells include interstitial cells of Cajal like cells (ICLC), pericytes, as well as the lymphatic muscle (LMCs) cells themselves. Here we tested the extent to which these cell types are invested into the mouse cLV wall and if any cell type exhibited morphological and functional processes characteristic of pacemaker cells: a contiguous network; spontaneous Ca2+ transients; and depolarization-induced propagated contractions. We employed inducible Cre (iCre) mouse models routinely used to target these specific cell populations including: c-kitCreERT2 to target ICLC; PdgfrβCreERT2 to target pericytes; PdgfrαCreER™ to target CD34+ adventitial fibroblast-like cells or ICLC; and Myh11CreERT2 to target LMCs. These specific inducible Cre lines were crossed to the fluorescent reporter ROSA26mT/mG, the genetically encoded Ca2+ sensor GCaMP6f, and the light-activated cation channel rhodopsin2 (ChR2). c-KitCreERT2 labeled both a sparse population of LECs and round adventitial cells that responded to the mast cell activator compound 48–80. PdgfrβCreERT2 drove recombination in both adventitial cells and LMCs, limiting its power to discriminate a pericyte specific population. PdgfrαCreER™ labeled a large population of interconnected, oak leaf-shaped cells primarily along the adventitial surface of the vessel. Titrated induction of the smooth muscle-specific Myh11CreERT2 revealed a LMC population with heterogeneous morphology. Only LMCs consistently, but heterogeneously, displayed spontaneous Ca2+ events during the diastolic period of the contraction cycle, and whose frequency was modulated in a pressure-dependent manner. Optogenetic depolarization through the expression of ChR2 by Myh11CreERT2, but not PdgfrαCreER™ or c-KitCreERT2, resulted in a propagated contraction. These findings support the conclusion that LMCs, or a subset of LMCs, are responsible for mouse cLV pacemaking.

al., 1977) and this function is significantly compromised in patients suffering from lymphedema, 89 whose cLVs typically display weak and irregular or entirely absent contractile activity 90 (Olszewski, 2002). Ex vivo studies, in which the intraluminal pressure can be precisely 91 controlled, have refined our understanding of the pressure-dependent regulation of 92 contraction frequency (Benoit et al., 1989;Gashev et al., 2004), with some mouse cLVs 93 displaying a 10-fold increase in contraction frequency over a 10 cmH2O pressure gradient 94 ( their simplified architecture, compared to larger mammals, in combination with the genetic 134 tools developed for the mouse model, allowed us to test for a fundamental pacemaker cell in 135 the cLV. In this study we utilized multiple genetic mouse models, immunofluorescence imaging, 136 Ca 2+ imaging, and optogenetic light-activated depolarization to both visualize and test the 137 functional aspects of putative pacemaker cells. We did not observe a significant or contiguous 138 cKit + cell population along the vessels. PdgfrαCreER TM was able to consistently delineate a 139 significant population of interconnected adventitial cells that were also CD34 + . However, an 140 absence of Ca 2+ events in phase with the contractile activity and an inability to consistently 141 elicit a contraction upon photo-stimulated depolarization provides functional evidence against 142 a role for either PDGFRα + or cKit + adventitial cells as the pacemaker cell. In contrast, MYH11 + 143 LMCs exhibited diastolic Ca 2+ events that, while asynchronous, were dynamically modulated by 144 pressure and could elicit a propagated contraction after optogenetic stimulation of LMCs 145 expressing ChR2.

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Results 148 Methylene Blue Staining Reveals adventitial Cells in Murine cLVs 149 Methylene blue staining was used to identify an ICLC population in the human lymphatic 150 thoracic duct (Briggs Boedtkjer et al., 2013). In our isolated and cleaned IALVs ,methylene blue 151 stained a significant number of cells along the length of the vessel. The methylene blue + cells 152 had variable density along the length of the vessel and stained cells exhibited different patterns 153 of morphology and location ( Figure 1A-C). A significant portion of the stained cells resembled 154 lymphatic vessel-associated macrophages with an elongated spindle shape, while other cells 155 were smaller and circular ( Figure 1D-F). Methylene blue also appeared to stain mast cells as 156 there were large ovoid cells on the adventitia of the vessel with intracellular granules. In 157 addition, methylene blue stained a minor population of cells that exhibited long and thin axon-158 like extensions which appeared to have a slight helical orientation, with a small central body 159 and nucleus ( Figure 1C). None of these cell populations were aligned with the longitudinal axis 160 of the vessel that would permit efficient coupling or regulation across the circumferential LMCs 161 required for coordinated propagation along the length of the vessel. 162 163 Immunofluorescence Imaging of IALVs stained for ICLC, LEC, and LMC Markers 164 We first stained IALVs for the putative telocyte/ICLC markers cKit, CD34, and the intermediate revealed a striking population of cells that were seemingly contiguous along the length of the 173 vessel. These CD34 + cells generally had multiple lobular processes and a "oak leaf" like 174 appearance, characteristic of fibroblasts, while some contained short, thin dendrite-like 175 extensions ( Figure 2C, G, K). CD34 + cells were negative for desmin ( Figure 2H), which stained 176 the circumferential LMCs ( Figure 2F; note that the largely non-circumferential cell organization 177 in this region is typical for a lymphatic endothelial valve site). Furthermore, the CD34 + cells and 178 cKit + stained separate populations ( Figure 2D, L). Vimentin stained lymphatic endothelial cells 179 (LECs) which exhibited a horizontal cobblestone morphology in parallel with the vessel axis 180 ( Figure 2E, I), while also co-labeling the majority of the CD34 + cells ( Figure 2H) and cKit + cells 181 ( Figure 2L). Videos of the half vessel z-stacks are provided (Supplemental Movies 1-3 for Figure  182 2D, H, and L respectively).

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During the imaging of mouse IALVs for these markers, we also observed that the lymphatic 206 secondary endothelial valves were populated by elongated cells that stretched the length of the 207 valve leaflet and were positive for CD34, PDGFRα, and PDGFRβ, with varying intensities, and 208 found within both leaflets of the valve ( Figure 3V,W) as determined from max projections of a 209 z-stack encompassing only the "z slices" within the lumen of the IALV. These cells had long, thin 210 extensions that were branched along with apparent dendrite extensions with a morphology 211 that closely resembled those described of pericytes or telocytes  Pellegrini, 2010) ( Figure 3V,W). PDGFRα + or CD34 + cells with this morphology were only 213 observed in the valve leaflets and thus seemed insufficient to regulate pacemaking as normal 214 contractions are observed in cLVs without secondary valves (not shown). The z-stacks 215 demonstrating these valve-located "telocyte" shaped cells ( Figure 3V,W) are provided as 216 Supplemental Movies 4 and 5. 217 The vast majority of the PDGFRα + cells were located in the adventitial layer Figure 4A-D, 218 which varied between 2-3 PDGFRα + cells thick ( Figure 4E) for this particular mouse lymphatic 219 collecting vessel. Under this layer, we observed only a single layer of largely circumferential 220 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted August 26, 2023. ; https://doi.org/10.1101/2023.08.24.554619 doi: bioRxiv preprint LMCs stained by MYH11 ( Figure 4B) sitting atop a single layer of CD31 + LECs ( Figure 4A). We 221 also observed occasional PDGFRα + cells or their extensions located in the sub-endothelial space 222 (Figure 4 E', E") positioned between the LECs and the LMCs. 223 We next determined the degree of colocalization between the CD34 and PDGFRα signal 224 given the significant overlap. Colocalization analysis of PDGFRα ( Figure 5A) and CD34 ( Figure  225 5B) and their colocalization ( Figure 5C) was determined with the FIJI BIOP-JACoP tool. The 226 Pearson's coefficient was 0.83 ( Figure 5 D) and Mander's coefficient of overlap 0.80 was for the 227 PDGFRα + signal and 0.87 for the CD34 signal ( Figure 5E). This high degree of colocalization CD34 228 and PDGFRα signal informed our use of the commercially available PdgfrαCreER TM mouse model 229 to target these cells.

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Use of iCre-Mediated Recombination of Rosa26mT/mG to delineate and characterize specific 232 IALV cell types 233 After confirming the presence of Vimentin + , cKit + , and CD34 + PDGFRα + positive cells within the 234 mouse IALV, we sought to further investigate these cell populations by using constitutive and 235 inducible Cre recombinase expressing mouse lines. IALVs from the constitutively active 236 PdgfrαCre-ROSA26mTmG and Ng2Cre-ROSA26mTmG mice had GFP fluorescence in the 237 majority of LMCs as well as in the fibroblast-shaped cells found within the IALV wall ( Figure 6  238 A,B). While informative of expression of the LMC progenitor cells, neither constitutive Cre 239 would be useful in delineating cell types. In contrast to the constitutively active PdgfrαCre, the 240 tamoxifen inducible PdgfrαCre TM line drove significant recombination in only the fibroblast-241 shaped cells previously stained with CD34 and PDGFRα but not LMCs or LECs ( Figure 6C).

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PdgfrβCreER T2 , commonly used to label pericytes, drove recombination in both a minor 243 population of the LMCs and the fibroblast-shaped cells. cKitCreER T2 , which capably drives 244 recombination in the ICC of the GI (Baker et al., 2016), drove recombination only in a small 245 population of irregularly-spaced large ovoid cells on the surface of the IALV ( Figure 6E), largely 246 corresponding with mast cell labeling, although recombination in 1 or 2 LECs could occasionally 247 be detected (not shown). Finally, Myh11CreER T2 drove recombination in nearly all LMCs which 248 were largely circumferentially oriented with dendrite-like, cell-cell contacts visible between 249 them and without significant GFP fluorescence in either LECs or the fibroblast-shaped CD34 + 250 PDGFRα + cell population ( Figure 6F). Additionally, some LMCs maintained the bipolar shape but 251 had multiple extensions forming a "Y" shape in which an adjacent LMC typically filled the inner 252 void. A very minor population of recombined cells in the Myh11CreER T2 -ROSA26mTmG IALVs 253 were smaller and irregularly patterned with multiple fine axon-like projections or highly-254 dendritic ruffled edges ( Figure 6F).

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To complement the morphological and cell density findings obtained with confocal 256 microscopy, we digested IALVs from the iCre-ROSA26mTmG lines, and the Prox1-eGFP line as a 257 control, into single cell suspensions and sorted the respective GFP + populations ( Figure 6G-J) for 258 RT-PCR profiling ( Figure 6K). We first focused on determining the molecular fidelity of the 259 sorted cells based on the gene promoters used to drive each "iCre" model to discern cellular 260 overlap. In agreement with the confocal images, sorted GFP + cells from PdgfrβCreER T2 -261 ROSA26mT/mG IALVs expressed PDGFRβ but also MYH11 and PDGFRα. In contrast, GFP-sorted 262 cells from PdgfrαCreER TM IALVs expressed PDGFRα and PDGFRβ, but with no detectable 263 expression of MYH11. Conversely, GFP + cells from sorted Myh11CreER T2 -ROSA26mTmG IALVs 264 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made We further profiled each population of sorted cells with RT-PCR for other common 268 markers for endothelial cells, muscle cells, and pericytes. Similar to the Prox1 results ( Figure  269 6K), endothelial nitric oxide synthase (eNOS) expression was observed only in the Prox1-eGFP 270 sorted cells, which also expressed vimentin and MCAM and had weak but detectable signal for 271 CD34 ( Figure 7A). Myh11CreER T2 sorted cells showed expression of smooth muscle actin 272 (Acta2), the alpha subunit of the L-type voltage gated Ca 2+ channel Cav1.2, desmin, MCAM, and 273 vimentin ( Figure 7B). In addition to the genes expressed under Myh11CreER T2 recombination, 274 Cdh5, CD34, and Cspg4 (Ng2) were detected in cells sorted in PdgfrβCreER T2 IALVs ( Figure 7C).

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As expected, the GFP + cells sorted from PdgfrαCreER TM IALVs expressed mRNA for CD34, Cspg4, 276 and vimentin, but not desmin, smooth muscle actin, nor the pericyte marker MCAM ( Figure  277 7D). The alpha subunit of the voltage gated Ca 2+ channel was positive in cells sorted from both 278 PDGFRα, PDGFRβ,and Myh11CreER T2 IALVs, but was also observed in the PdgfrαCreER TM IALVs 279 without any evidence that MYH11 expressing muscle cells contaminated the latter. These 280 findings confirmed the separate cell populations achieved with PdgfrαCre TM and Myh11CreER T2 281 mediated recombination, at least as it pertains to ROSA26mTmG. We followed up the 282 identification of Cav1.2 expression in the PdgfrαCreER TM sorted cell population by assessing the 283 expression of other genes involved in either pacemaking (Ano1) or electrical conduction (Cx45) 284 of IALVs ( Figure 7E). Intriguingly, expression of both Ano1 and Cx45 was observed in the 285 PdgfrαCreER TM sorted cells, which were further confirmed to lack either endothelial or 286 hematopoietic contamination as we did not detect expression of the endothelial marker CD31 287 or the hematopoietic marker CD45.

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Inducible Deletion of Either Cav1.2, Ano1, and Cx45 with PdgfrαCreER TM Did Not Affect cLV 290 Pacemaking 291 The expression of the genes critically involved in cLV function-Cav1.2, Ano1, and 292 Cx45-n the PdgfrαCreER TM -ROSA26mTmG purified cells prompted us to generate 293 PdgfrαCreER TM -Ano1fl/fl, PdgfrαCreER TM -Cx45fl/fl, and PdgfrαCreER TM -Cav1.2fl/fl mice for 294 functional testing. We isolated popliteal cLVs and tested their pacemaker and contractile 295 activity in response to physiological pressures over the range 0.5-10 cmH2O, under normal 296 conditions. However, we did not detect any significant difference in pacemaking nor contractile 297 function of popliteal cLVs studied from PdgfrαCreER TM -Ano1fl/fl ( Figure 8A-F) or 298 PdgfrαCreER TM -Cx45fl/fl (Figure9A-F), compared to their respective Ano1fl/f and Cx45fl/fl 299 controls. PdgfrαCreER TM -Cav1.2fl/fl mice had no statistically significant differences in 300 normalized contraction amplitude ( Figure 10A), contraction frequency ( Figure 10C), fractional 301 pump flow ( Figure 10D), end diastolic diameter ( Figure 10E). However, we noted a mild but 302 statistically significant increase in ejection fraction at the lowest pressure, 0.5 cmH2O. Vessels 303 isolated from PdgfrαCreER TM -Cav1.2fl/fl mice also had a statistically significant increase in 304 vessel tone ( Figure 10F) noted at the 2-way ANOVA level although we did not resolve 305 significance at any specific pressure with this sample size. Nonetheless, despite the presence of 306 transcript for these critical genes, PdgfrαCreER TM mediated deletion failed to recapitulate 307 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made Despite the lack of cLV pacemaking deficits in the PdgfrαCreER TM genetic knockout lines, we 312 were curious to discern further insight into the role or function of the PDGFRα + CD34 + cells 313 which comprise a significant portion of the lymphatic cLV wall. The PdgfrαCreER TM recombined 314 cells exhibited expression of Krüppel-like factor 4 (Klf4), stem cell antigen 1 (Sca1, also referred 315 to as Ly6a), Gli1, CD29, CD105, and CD44 ( Figure 11A, B). To Figure 11C). Similarly, LECs sorted from Prox1-eGFP IALVs 320 were positive for Klf4, weak for Sca1, and positive for CD34 but negative for GLi1 and PDGFRα 321 Gli1. In contrast, PdgfrαCreER TM -ROSA26mTmG recombined cells were positive for all markers 322 as were the unrecombined population (tdTomato + ) cells in the Myh11CreER T2 -ROSA26mTmG 323 IALVs ( Figure 11D). We performed immunofluorescence staining for one of these multipotent 324 markers, Sca1 ( Figure 11E, I) while counter staining for LMCs, with SMA or MYH11 ( Figure 11F, 325 J), and the adventitial cells with PDGFRα ( Figure 11G, K). The morphology and staining pattern 326 of Sca1 overlapped significantly with PDGFRα staining and not the LMC staining ( Figure 11H, L, 327 Supplemental Movie 6).

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Optogenetic Stimulation of iCre-driven Channel Rhodopsin 2 to Induce Test Light-Stimulated 330 Depolarization Induced Lymphatic Contraction 331 We next used optogenetic methods to test whether the cell populations recombined by either 332 cKitCreER T2 , PdgfrαCreER TM , or Myh11CreER T2 could elicit a coordinated contraction. The ChR2-333 tdTomato construct appeared more sensitive to recombination than ROSA26mTmG, in some 334 cases resulting in LMC expression of ChR2-tdTomato in PdgfrαCreER TM and CKitCreER T2 vessels.

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Care was taken to image the vessel for tdTomato ( Figure 12A,C,E) prior to stimulation at their 336 respective sites under brightfield conditions for diameter tracking ( Figure 12B,D,F) to ensure 337 fidelity of the cell types and morphologies observed in Figure 3. As with ROSA26mTmG, 338 CKitCreER T2 drove the ChR2-tdTomato expression primarily in large ovoid cells found on the 339 adventitia of the vessel. Photo-stimulation of these cells did not initiate coordinated 340 contractions ( Figure 12G-J,S). Similarly, photo-stimulation of ChR2-tdTomato expressing cells 341 driven by PdgfrαCreER TM failed to initiate a coordinated contraction ( Figure 12K-N, T). In 342 contrast, photo-stimulation of LMCs, using Myh11CreERT2 to express Chr2-tdTomato, resulted 343 in a propagated contraction in the popliteal vessel ( Figure 12O-R, U). In total, only 3.25% of 344 photo-stimulations of cKitCreER T2 -ChR2-TdTomato and 3.03% of photo-stimulations of 345 PdgfrαCreER TM -ChR2-tdTomato were associated with a contraction, while 88.5% of photo-346 stimulations of Myh11CreER T2 -ChR2-tdTomato photo-stimulations induced contractions ( Figure  347 12V). The low percentages of optogenetic firing of contractions observed in PdgfrαCreER TM -348 ChR2-tdTomato and cKitCreER T2 -ChR2-TdTomato vessels are likely due to the happenstance of 349 spontaneous contractions occurring during the time and proximity of optogenetic stimulation.

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As mast cells are not ascribed any tissue specific pacemaking behavior, the similar percentages 351 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made We imaged IALVs from cKitCreER T2 -GCaMp6f mice, which primarily resulted in expression of 358 GCaMp6f in the large ovoid cells in the adventitia ( Figure 13A), although we occasionally 359 observed GCaMP6f expression in both LEC and LMCs ( Figure 13A) as depicted in the maximum 360 projection of the acquisition period (Supplemental Movie 10) and the spatio-temporal maps 361 (STMS). The aberrant expressions of GCaMP6f in cells that demonstrated the typical 362 cobblestone morphology of LECs or the circumferential LMCs that exhibited Ca 2+ flashes and 363 diastolic Ca 2+ transients ( Figure 13D,E green arrows) prior to contraction ( Figure 13D,E) were 364 not included in the cKitCreER T2 -GCaMp6f analysis. Of the 39 cKitCreER T2 -GCaMp6f cells 365 analyzed, only 1 cKitCreER T2 -GCaMP6f cell exhibited a spontaneous Ca 2+ transient during the 366 recording period ( Figure 13B,C Cell 7). However, the Ca 2+ transient in that cell did not align 367 temporally with the "Ca 2+ flash" of the LMC with incidental GCaMp6f expression ( Figure  368 13C,D). Despite the lack of Ca 2+ transients under the baseline conditions throughout the IALV 369 contraction cycle, many cKitCreER T2 -GCaMP6f cells exhibited a robust and prolonged Ca 2+ event 370 in response to stimulation with the mast cell activator compound 48-80 ( Figure 13F, G, H). 371 Notably, the Ca 2+ events in the ovoid cells elicited by administration of compound 48-80 did not 372 acutely alter the LMC Ca 2+ activity ( Figure 13I,J). Similar to cKitCreER T2 -GCaMp6f, the majority of 373 PDFRaCreER TM -GCaMP6f expressing cells also largely lacked Ca 2+ transients and also resulted in 374 incidental LMC GCaMP6f expression ( Figure 14B, Supplemental Movie 11). Some cells exhibited 375 high basal Ca 2+ ( Figure 14A,D) sustained throughout the recording, but oscillations were not 376 observed ( Figure 14B,C). In contrast, spurious GCaMP6f expression in a circumferentially 377 oriented LMC displayed Ca 2+ flashes associated with contraction ( Figure 14B,C). Of the 21 378 PDGFRα-GCaMP6f cells assessed, only 3 exhibited Ca 2+ transients which were singular events 379 with limited spatial spread within the 20 sec imaging period ( Figure 14E,F). The lack of either 380 global or consistent Ca 2+ transients within either cKitCreER T2 -GCaMP6f or PdgfrαCreER TM -381 GCaMP6f IALVs was in stark contrast to Ca 2+ imaging of Myh11CreER T2 -GCaMP6f IALVs. 382 Myh11CreER T2 drove GCaMp6f expression in the circumferential LMCs ( Figure 15A), which had 383 global and nearly synchronous Ca 2+ flashes in 100% of the analyzed cells ( Figure 15B, C). 384 Additionally, non-synchronous stochastic and localized Ca 2+ transients during diastole were 385 commonly observed in the LMCs ( Figure 15D, E, Supplemental Movie 12 (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted August 26, 2023. ; https://doi.org/10.1101/2023.08.24.554619 doi: bioRxiv preprint were studied in the presence of nifedipine, which blocks the calcium flashes but not local 396 calcium transients at intraluminal pressures of 0.5 -5 cmH2O ( Figure 16A). As intra-luminal 397 pressure was increased, there was a marked increase in the occurrence of Ca 2+ transients 398 ( Figure 16B, Supplemental Movies 13-15). By converting raw Ca 2+ transients for particle 399 analysis (PTCLs), we generated activity maps of Ca 2+ PTCL activity ( Figure 16C) and determined 400 PTCL area ( Figure 16D) and frequency at each pressure ( Figure 16E). The maps show that as 401 pressure increased, the activity of PTCLs across the vessel also increased (as evident by the 402 increase in PTCL area activation). Across 11 experiments, the area of the field of view activated 403 by PTCLs/frame increased from 73.2 ± 17.7 mm 2 /frame at 0.5 cmH20 to 108.6 ± 20.5 404 mm 2 /frame at 2 cm H20 and further enhanced to 139.2 ± 26.9 mm 2 /frame at 5 cm H2O ( Figure  405 16F). The number of PTCLs per frame also increased with pressure, from 2.9 ± 0.4 at 0.5 cmH20 406 to 4.1 ± 0.5 and 5.2 ± 0.6 PTCL/frame at 2 and 5 cmH20 respectively ( Figure 16E). 407 408 Discussion 409 The In many smooth muscle organs, regulation of a coordinated contraction is a complex and 435 multicellular phenomenon. Multiple cell types integrate physical and biological information into 436 electrical activity to be transmitted to the force-producing smooth muscle cells, sometimes 437 across great distances relative to cell size, to regulate calcium influx by voltage dependent 438 calcium channels required for contraction. The intestine is one such documented tissue in 439 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made junctions and, as stated above, we detected Cx45 expression in the PdgfrαCreER TM sorted cells. 569 However, we did not detect any impairment in pacemaking, nor were contraction conduction 570 speed deficits or multiple pacemakers noted in the PdgfrαCreER TM -Cx45fl/fl popliteal cLVs, in 571 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made in contractions that were indistinguishable from the spontaneously occurring contractions. 583 These results give functional credence to the lack of hetero-cellular coupling that was 584 previously reported . Just as critically, they also highlight the regenerative nature of the 585 lymphatic muscle action potential as local depolarization was sufficient to drive a coordinated 586 contraction along the vessel and that a single or few LMCs reaching threshold for AP initiation 587 are sufficient to drive the conducted activity observed at the tissue level.

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Conclusions 590 Our present findings lend further support to the hypothesis that the LMCs are intrinsic and influx pathways, and the contributions of Ca 2+ sensitive ion channels need to be identified 597 to develop sophisticated in silico models and identify potential therapeutic targets to rescue 598 lymphatic pacemaking in lymphedema patients (Olszewski, 2002(Olszewski, , 2008. 599 600 Limitations 601 One assumption underlying our conclusions is that there is a conserved fundamental 602 pacemaking pathway in lymphatic muscle pacemaking across species, specifically pertaining to (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted August 26, 2023. ; https://doi.org/10.1101/2023.08.24.554619 doi: bioRxiv preprint Our data demonstrates that limited staining of a few cell markers alone is insufficient to identify 615 discrete cell populations in the murine cLVs. Additionally, mRNA expression does not equal 616 protein translation nor guarantee specific function as we did not detect endothelial CD34 with 617 immunofluorescence despite detecting transcript; additionally, PdgfrαCreER TM mediated 618 deletion of Ano1, Cx45, or Cav1.2 had no effect on cLV pacemaking. Hence, further 619 experimentation is also required to fully characterize expression of multipotent cell markers 620 and function of CD34 + PDGFRα + Sca1 + cells invested within the murine cLVs, although this was 621 beyond the scope of this study assessing pacemaker identity. Tangentially PdgfrβCreER T2 -ROSA26mT/mG, Myh11CreER T2 -ROSA26mT/mG, and cKitCreER T2 -ROSA26mT/mG 667 mice, respectively. The resulting iCre-ROSA26mT/mG mice were induced with tamoxifen 2-4 668 weeks after weaning. Tamoxifen induction was performed via consecutive 100 μL i.p. injections 669 of tamoxifen ranging from 1 to 5 days at concentration ranging from 0.2 -10 mg/mL in safflower 670 oil, using a titrated induction protocol to determine the extent of recombination in specific cell 671 populations. We used our maximal induction protocol, 100 μL of tamoxifen at 10 mg/mL over 5 672 consecutive days, for cKitCreER T2 -GCaMP6f, Myh11CreER T2 -GCaMP6f, and PdgfrαCreER TM -673 GCaMP6f mice. Due to the paucity of recombined cells in the cKitCreER T2 -ROSA26mT/mG 674 reporter mice, we used our maximal tamoxifen induction protocol for cKitCreER T2 -ChR2 mice as 675 this still resulted in the ability to excite single recombined cells. Myh11CreER T2 -ChR2/tdTomato 676 mice were induced with one 100 μL i.p. injection of tamoxifen at 0.2 mg/mL while PdgfrαCreER TM -677 ChR2/tdTomato were induced with 1 injection at 0.4 mg/mL tamoxifen to get mosaic induction 678 sufficient for single cell stimulation. All mice, regardless of duration, were given 2 weeks to 679 recover following tamoxifen injection.

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Lymphatic Vessel Isolation-We utilized both popliteal and inguinal-axillary lymphatic collecting 682 vessels (IALVs) in this study, which were isolated as described previously . In 683 brief, mice were anaesthetized with a cocktail of 100/10 mg/mL) ketamine/xylazine mg/mL and 684 shaved along the flank or the legs for IALVs and popliteal cLVs respectively. The IALV (also 685 referred to as the flank cLV) is located adjacent to the thoracoepigastric vein and connects the 686 inguinal and axillary lymph node. A cut was made along the dorsal midline and the skin retracted 687 and pinned out to reveal the thoracoepigastric vascular bed. The thoracoepigastric vascular bed 688 and connected perivascular adipose containing the IALVs vessel was dissected out and pinned 689 onto a Sylgard coated dish in Krebs buffer. Popliteal lymphatic vessels were exposed through a 690 superficial incision in the leg, removed and transferred to the Krebs-albumin filled dissection 691 chamber. After removal, the vessel was carefully cleaned of adipocytes and excess matrix using PdgfrαCreER TM -Cav1.2fl/fl mice. Theses mice and their respective fl/fl controls were injected 700 with tamoxifen as described above for 5 days and given two weeks to recover. The popliteal 701 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted August 26, 2023. ; https://doi.org/10.1101/2023.08.24.554619 doi: bioRxiv preprint vessels were isolated, cleaned, and prepared for isobaric contractile tests as previously 702 reported (Davis et al., 2023). Once equilibrated, inner diameter was tracked over a physiological 703 pressure range (stepped from 3 to 2, 1, 0.5, 3, 5, 8, and 10 cmH2O) with 2min of recording at 704 each pressure. Following the pressure step protocol the vessels were equilibrated in with Ca 2+ -705 free Krebs buffer (3mM EGTA) and diameter at each pressure recorded under passive 706 conditions. The contractile parameters end diastolic diameter (EDD), end systolic diameter 707 (ESD), and contraction frequency (FREQ) were recorded with a custom LabVIEW program and 708 the following contractile parameters assessed: Methylene Blue-Isolated IALVs sections were transferred into a Krebs-BSA buffer filled 3-mL 716 observation chamber, with a cover slip bottom, and cannulated onto two glass micropipettes (30-717 80 μm, outer diameter) held in place by pipette holders on a Burg-style V-track mounting system. 718 The pipette holders were attached to a 3-way valve stop cock with polyethylene tubing filled with 719 Krebs-BSA buffer. Vessels were pressurized to approximately 5 cmH2O by raising the 3-way valve 720 and the vessels were stretched to remove any slack. For methylene blue staining, IALVs from wild 721 type C57Bl6 mice were stained with 50 µM methylene blue in Krebs-BSA buffer for two hours at 722 room temperature and covered in foil to limit light induced phototoxicity. After the staining 723 period, the vessel chambers were washed three times with Ca 2+ free PSS to remove methylene 724 blue. Brightfield images and manual Z-stack videos were collected on an inverted Leica DMi1 4X 725 or 20X air objective, or a Leica DMi8 with a 25X water objective or an inverted DMi8 using a Leica 726 Flexacam C1 color camera for image acquisition. Some Methylene blue images were also 727 collected using a color Nikon DS-Fi3 camera. The collected z-stacks were analyzed using Image J 728 and the "Stack Focuser" plugin (https://imagej.nih.gov/ij/plugins/stack-focuser.html). To 729 accentuate the methylene blue stained cells, the color image stack was split into red, green, and 730 blue channel stacks. The blue channel stack was then divided by the green channel stack using 731 the "Image Calculator" function. The resulting 32-bit image was then converted into 16-bit image 732 to permit the use of the Stack Focuser plugin with the 'n kernel value' set to 11. 733 734 Fluorescence Confocal Imaging-IALVs vessels from each respective iCre-ROSA26mT/mG mouse 735 were prepared in a similar manner (excluding the addition of methylene blue). We performed 736 confocal imaging to acquire z-stacks of 7-10 overlapping regions of interests to allow for manual 737 stitching, with 1 μM z-steps (20Χ) or 0.5 μM steps at 40X. We imaged through to the midpoint of 738 the vessel except when imaging the valve interstitial cells, in which case the entire vessel was 739 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted August 26, 2023. ; https://doi.org/10.1101/2023.08.24.554619 doi: bioRxiv preprint imaged. Max projections were made using FIJI. Following live imaging, the vessels were 740 pressurized to 5 cmH2O and fixed with 4% paraformaldehyde for 30 min at room temperature. 741 IALVs were then washed with PBS containing 0.1% Triton X-100 (PBST) 3 times and blocked for a 742 minimum of 2 hr with Blockaid® (B-10710, ThermoFisher Scientific). IALVs were then stained with 743 the corresponding primary antibodies in BlockAid® Solution: anti-smooth muscle actin (SMA) were re-cannulated and pressurized for imaging using the aforementioned spinning disk confocal 752 and Hamamatsu Orca Flash4 camera using a 20X air objective (Olympus UplanApo, 0.75) or 40X 753 (Olympus UApo A340, 1.15) water objective. Images were taken as described above, and the 754 resulting stacks were turned into a max projection using FIJI. Ca 2+ Imaging and Analysis in IALVs Over the Contraction Cycle-Background noise was 823 determined by using the histogram feature of FIJI in a rectangle in a region of the field of view 824 without sample. This value was subtracted from the entire field of view. In some cases, the vessel 825 movement due to contraction was offset with video stabilization with the FIJI plugin Image 826 Stabilizer. A max projection was used to create non-overlapping ROIs of GCaMP6f + cells for each 827 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted August 26, 2023. ; https://doi.org/10.1101/2023.08.24.554619 doi: bioRxiv preprint iCre-GCaMp6f IALV. From these cell ROIs, the "reslice z" function was used to create a pseudo-828 linescan STMs which were divided by their baseline values to obtain F/F0 values for each 829 individual cell. At least 3 cells, except in the case of 1 cKitCreER T2 -GCaMp6f IALV, in which only 830 two cells were observed, were analyzed in this manner for each vessel segment. Max projections 831 of the image stack were then used to create non-overlapping cell masks of 3-5 muscle cells per 832 field of view of one vessel. Ca 2+ traces for those cells contained 5-10 contraction cycles and Ca 2+ 833 transients and were characterized for peak intensity (expressed as a baseline-referenced ratio, 834 F/F0), frequency, and duration in seconds.

836
Analysis of Subcellular Ca 2+ Transients in Myh11CreER T2 -GCaMP6f IALVs For Myh11CreER T2 -We 837 performed Ca 2+ imaging as above in the presence of 1 µM nifedipine to stop the "Ca 2+ flashes" 838 associated with action potentials  and focus on the subcellular activity at 3 839 different experimental pressures of 0.5, 2, and 5 cmH2O. For this protocol, we used a particle 840 analysis approach to analyze all Ca 2+ transients in the field of view. Ca 2+ transients in intact vessels 841 were quantified by particle analysis as previously described ( PTCLs <10 mm 2 were rejected to facilitate the removal of noise and then the total PTCL area and 847 PTCL count could be tabulated for each recording.

849
Light Activation of ChR2 to stimulate Popliteal Collecting Lymphatic Vessel Contractions.

850
As the IALV has a nearly continuous contractile cycle, we utilized the popliteal vessel for its much 851 slower contraction frequency in the experiments testing our ability to trigger a propagated 852 contraction upon stimulation of the enforced expression of ChR2. Popliteal vessels were isolated 853 from cKitCreER T2 -ChR2/tdTomato, PdgfrαCreER TM -ChR2/tdTomato, or Myh11CreER T2 -854 ChR2/tdTomato mice as previously described (Scallan and Davis, 2013), although we intentionally 855 retained some connective tissue and adipose tissue to ensure we had a sufficient population of 856 recombined cells to test in the adventitia layer of the vessel. Contractions were allowed to 857 stabilize over a 30-min equilibration period with pressure set to 3 cmH2O. If basal contraction 858 frequency was too high, we applied pinacidil to the bath in 100 nM increments, without 859 exceeding 600 nM, to further slow contraction frequency to around 6 contractions per minute. 860 Pinacidil at sub 1 µM doses can slow contraction frequency without causing overt 861 hyperpolarization of membrane potential (Davis et al., 2020). Supplemental 100 nM doses of 862 pinacidil were applied throughout the experiment to maintain a spontaneous contraction 863 frequency below 6 per minute to allow ample diastolic time for ChR2 stimulation. Throughout 864 this protocol the popliteal was allowed to contract spontaneously to ensure we had not overly 865 inhibited action potentials by the pacemaking cells with pinacidil. Occasionally spontaneous 866 contractions occurred just prior to contractions and could result in a potential false positive so 867 we performed multiple stimulations over a period of 5 -10 min, typically waiting at least 3 s after 868 any spontaneous contraction before stimulating. Care was made to align the light fiber in such a 869 way that only part of the vessel would be directly illuminated and so target cells of interest would 870 be directly activated by 473 nm light using a Laser diode (Doric LD Fiber Light Source, Quebec, 871 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted August 26, 2023. ; https://doi.org/10.1101/2023.08.24.554619 doi: bioRxiv preprint Canada), through an optical probe with a 10-µm tip (Doric, OPT_200_0.22.010). To further limit 872 the excitation field, the optical probe was coated with black acrylic paint using an eyelash brush 873 so that the uncoated opening was ~2-3 µm. With the probe positioned within 5 µm of one side 874 of the vessel wall, the spread of light covered an area ~10-100 µm wide on the back side of the 875 vessel (depending on the diode amplitude setting). Light pulses, 200 ms in length, were triggered 876 by a Grass S9 stimulator (Harvard Apparatus, Holliston, MA) connected to the external TTL input 877 of the laser diode. Pulse amplitude was adjusted between 40-90 mA using the Laser Diode 878 Module Driver (Doric). A contraction was considered to be triggered if it occurred within 50ms of 879 stimulation. We performed photo-stimulation from 2-4 sites within each vessel, with 6-14 880 stimulations per site. If a photo-stimulation was triggered incidentally after the initiation of a 881 "spontaneous contraction" it was discarded from the analysis. For Myh11CreER T2 -ChR2-882 tdTomato 6 vessels from 3 separate mice were tested. For PdgfrαCreER TM -ChR2-tdTomato 6 883 vessels from 4 separate mice were tested with a max of two vessels per mouse. For cKitCreER T2 -884 ChR2-tdTomato 7 vessels from 3 separate mice were assessed. Diameter was recorded to align 885 photo-activation with the contraction cycle in a custom Labview program.

898
Statistical Tests Statistical differences in the isobaric contractile tests for popliteal cLVs isolated 899 from PdgfrαCreER TM -Ano1fl/fl, PdgfrαCreER TM -Cx45fl/fl, and PdgfrαCreER TM -Cav1.2fl/fl mice 900 over the various contractile parameters were assessed via 1) two-way ANOVAs with Tukey's 901 multiple comparison tests data performed using Prism9 (Graphpad). Data are plotted as mean ± 902 SEM and significance determined at p < 0.05. We used a categorical Chi-squared statistical test 903 for the experiments assessing our ability to trigger a contraction with activation of ChR2 cells.

904
Ca 2+ PTCL area and frequency was compared using 1-way ANOVA with Tukey's post-hoc test.

905
Significance was determined at a p value of < 0.05. 906

907
Acknowledgements 908 We    Physiol. 301:H48-60. 1004 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted August 26, 2023. ; https://doi.org/10.1101/2023.08.24.554619 doi: bioRxiv preprint allows efficient inducible gene targeting in and ablation of mast cells. Eur J Immunol.
. CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted August 26, 2023. ; https://doi.org/10.1101/2023.08.24.554619 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted August 26, 2023.   PdgfrβCreER T2 -ROSA26mTmG (J) mice. Representative gels demonstrating RT-PCR products 1316 corresponding to the respective gene used in the promoter of the transgenes to drive GFP 1317 signal or Cre mediated recombination of ROSA26mTmG from each GFP + sorted population (K) 1318 to assess fidelity. Images are representative of IALVs from at least 3 separate mice. FACs and 1319 RT-PCR was repeated at least 3 times for each mouse. 1320 1321 1322 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made  (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted August 26, 2023. ; https://doi.org/10.1101/2023.08.24.554619 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted August 26, 2023. ; https://doi.org/10.1101/2023.08.24.554619 doi: bioRxiv preprint Figure 12 ChR2-Mediated Depolarization in LMCs Only Triggers Contraction 1374 Representative max projections of tdTomato-ChR2 signal in popliteal cLVs isolated from 1375 cKitCreER T2 -ChR2-tdTomato (A), PdgfrαCreER TM -ChR2-tdTomato (C), and Myh11CreER T2 -ChR2-1376 tdTomato (E) with their corresponding brightfield image (B, D, F) respectively. Time-lapse 1377 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted August 26, 2023. ; https://doi.org/10.1101/2023.08.24.554619 doi: bioRxiv preprint

Figure 13 cKitCreERT2 Drives GCaMP6f Expression in Primarily Mast Cells in Murine IALVs 1391
Representative max projection of GCaMP6f signal over time in an IALV isolated from a 1392 cKitCreER T2 -GCaMP6f mouse with ROI indicated around individual cells, primarily large ovoid 1393 cells, but also including a circumferential LMC (Cell10) and a horizontal LEC (Cell 11). Of cells 1-1394 9, only cell 7 had any Ca 2+ activity (red arrows) during the recording time as indicated by the 1395 STMs from each ROI (B) and their normalized F/F0 plots in (C). In contrast, the LMC in ROI 10 1396 had both rhythmic global Ca 2+ events (D) that spanned the cell axis (vertical axis) in the STM (E) 1397 in addition to localized Ca 2+ events intervening the time between global events (green arrows). 1398 Representative max projection of GCaMP6f signal over time after stimulation with C48-80 (F) 1399 with many large ovoid cells displaying long lasting global Ca 2+ events (G,H) while not 1400 immediately affecting the LMC Ca 2+ dynamics (I,J). 1401 1402 1403 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted August 26, 2023. shows the first diastolic period plotted in (D). 1426 1427 1428 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted August 26, 2023. ; https://doi.org/10.1101/2023.08.24.554619 doi: bioRxiv preprint 1429 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted August 26, 2023. ; https://doi.org/10.1101/2023.08.24.554619 doi: bioRxiv preprint