Developmental and transcriptional characterization of lymphatic muscle cells
Embargo Date
2026-03-17
OA Version
Citation
Abstract
Lymphatic vessel dysfunction has been associated with multiple diseases including obesity, inflammatory bowel disease and aging, and is directly responsible for lymphedema. The latter is a chronic and debilitating disease that does not have a cure nor effective treatment and is estimated to affect 120 million people worldwide. In individuals that acquire lymphedema after birth, the insults that damage lymphatic endothelium have been well characterized. However, recent studies suggest that pathological changes in lymphatic muscle cells (LMCs) may also contribute to lymphatic vessel impairment.LMCs are responsible for the unique contractile properties of lymphatic vessels and consequently, are one of the main drivers of lymph transport and lymphatic vessel function. LMCs perform coordinated contractions that have long been thought to be mediated by intra-cellular proteins of not only vascular smooth muscle cells (SMCs), but also visceral smooth muscle and striated muscle. Nevertheless, a thorough comparative expression analysis between LMCs and SMCs is missing in the literature, making it difficult to accurately characterize any differences. The understanding of the expression and developmental differences between LMCs and SMCs is critical to target LMCs surrounding collecting lymphatic vessels to improve lymph flow in patients with lymphedema.
In this work I first used conventional and conditional WT1 reporter mice to show LMC and SMC derivation from mesodermal progenitors in popliteal and mesenteric vessels during early embryonic development. I found that WT1 was expressed in early LMC progenitors but turned off in LMCs of adult mice. I found that WT1 was not re-expressed in adult LMCs upon lymphatic injury. Although not all LMC populations were derived from progenitors that expressed WT1, diphtheria toxin-mediated ablation of WT1-derived cells in the limb resulted in a significant decreased LMC coverage and decreased contractile function of the popliteal lymphatic vessel. To further study developmental heterogeneity in LMCs and SMCs, I used Nkx2.5 reporter mice and found that SMCs are not derived from cardiac progenitors. To reconcile differences in the literature that describe a hybrid LMC phenotype, I conducted single cell RNA sequencing (scRNAseq) of LMCs and SMCs from conventional WT1 reporter mice. scRNAseq showed that LMCs and SMCs derived from WT1+ progenitors were transcriptionally similar to those derived from other sources. Importantly, scRNAseq also showed transcriptional similarity between LMCs and venous SMCs, but both revealed unique markers compared to arteriolar SMCs, which were enriched in genes associated with cardiac contraction. Further lineage tracing using conditional Myh11 reporter mice showed that LMCs are not derived from mature SMCs during development, despite their transcriptional similarities. In conclusion, in this work I identified developmental heterogeneity among LMCs and SMCs from mesodermal sources. I also characterized their transcriptional profile to find the relevance of WT1 in vascular development and a previously unknown similarity between LMCs and venous SMCs. Future work will focus on characterization of additional lymphatic and blood vessels.
Description
2024