Primary cilia on LECs play a crucial role in lymphatic vasculature development and remodeling

Abstract

Primary cilia are non‐motile organelles which project from the cell surface and function to receive and process molecular signals. We have recently discovered that lymphatic endothelial cells (LECs) display primary cilia in vitro and in vivo in both developmental and inflammation/wound healing contexts. We have induced primary cilia formation in both immortalized mouse and primary adult human LECs by serum starvation protocols and detected them by immunofluorescence microscopy for ciliary markers ADP ribosylation factor like GTPase 13B (ARL13B) and acetylated alpha tubulin. We have used the mouse corneal suture model to create in vivo conditions of inflammation and wound recovery and found that primary cilia incidence both in LECs and globally in cornea is dependent on inflammatory status. Primary cilia assembly and transport of ciliary cargo is governed by a set of protein complexes made up of intraflagellar transport (IFT) proteins. To ascertain the role of intraflagellar transport in lymphatic development, we developed a conditional knockout (KO) mouse model of IFT20. At the gross level, E16.5 IFT20 KO mice embryos show hydrops, polydactyly, and craniofacial abnormalities. These embryos also possess a malformed, red blood cell‐filled lymphatic vessel (LV) phenotype. Using two photon microscopy of whole mount embryonic skin, we have found the IFT20 KO embryos to have higher lymphatic vessel density and larger LV diameter in comparison to negative littermate controls. We have found that the difference in LV diameter is not due to increased LEC proliferation. Differences in dorsal skin lymphatic vessel patterning across the midline have also been identified in the IFT20 KO. We have also confirmed decreased incidence of primary cilia in IFT20 KO embryonic skin using spinning disk confocal microscopy. Ongoing studies in these embryos are designed to characterize differences in smooth muscle actin coverage of collecting LVs, intercellular LEC junction protein expression and organization, and lymphatic valve formation and possible insufficiency. We have also generated a lymphatic‐specific conditional deletion mouse model of IFT20 loss and are working to understand whether the LV phenotypes present in the global IFT20 KO mouse are due to LEC‐intrinsic defects or to secondary effects on lymphatic vasculature due to defects in other cell types following loss of IFT20. The discovery of primary cilia on LECs may fundamentally change what is known about how extracellular signals are processed by lymphatic endothelium to direct development and remodeling of the lymphatic vasculature.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.