| The lymphatic system maintains fluid homeostasis by absorbing interstitial fluid and macromolecules and participates in immune regulation by transporting antigens and immune cells.The lymphatic system is a unidirectional conduit that returns lymph filtered by lymph nodes to the blood circulation.Previous studies have shown that lymphatic endothelial cells migrate as single cells to the proximal region of mouse lungs at embryonic day 11.5,and connect to form lymphatic vessels at embryonic day 14.5.However,little is known about the further development signatures of pulmonary lymphatic vessels.Pulmonary lymphatic vessels are essential for the clearance of fetal lung fluid,and the lungs of newborn mice lacking lymphatic vessels cannot establish normal respiratory function.Pulmonary lymphatic vessels are also involved in the maintenance of lung immune homeostasis in adult,and the lack of lymphatic function leads to chronic lung inflammation.However,the molecular mechanisms of pulmonary lymphatic function remain largely unknown.In this thesis,the development and function of pulmonary lymphatic vessels at different stages have been studied through single-cell RNA sequencing technology and mouse genetics.Through single-cell RNA sequencing of mouse pulmonary lymphatic endothelial cells at embryonic day 14.5,16.5,and 18.5,and postnatal day 0(4 hours)and day 62,we found four clusters of lymphatic endothelial cells.The first and second clusters mainly consist of embryonic cells.The third cluster mainly consists of postnatal day 0 cells.The fourth cluster mainly consists of postnatal day 62 cells.Four subpopulations were further analyzed by differential expressed gene comparison and functional enrichment.Cells of the first cluster highly express cell proliferation and ribosome-related genes.and cells of the second cluster highly express ribosome-related genes.The expression of ribosome-related genes indicates active protein translation.Combining single-cell RNA sequencing and experimental validations,we revealed that lymphatic vessels of the lung undergo active lymphangiogenesis from the embryonic stage to postnatal day 0,with increased branches and density,gradually covering the distal alveolar regions.Cells of the third cluster highly express a series of transcription factors,mainly of which are genes of the AP-1 family.Cells of the fourth cluster highly express genes regulating the immune system process.Ccl21 in lymphatic endothelial cells regulates the migration of T cells,dendritic cells,and B cells to the pulmonary lymphatic vessels under steady-state conditions.The pulmonary lymphatic endothelial cells in the neonatal stage perform fluid clearance function,and c-Jun is transiently expressed in the pulmonary lymphatic endothelial cells at this stage.After Jun knock-out in lymphatic endothelial cells,the mice showed respiratory defects and died at birth.Specifically,we demonstrated that lymphatic vessels at postnatal day 0 that lack c-Jun are not able to open the overlapping flaps between lymphatic endothelial cells that have been recognized to be sites for the intercellular entry of fluids and,thus,defective in fluid clearance.c-Jun as a transcription factor regulates Cdh13 expression in pulmonary lymphatic endothelial cells at birth.In vitro analysis in primary human pulmonary lymphatic endothelial cells suggested that c-JUN regulates cellcell junctions remodeling through CDH13.In summary,we have described and explored the characteristics and functions of pulmonary lymphatic endothelial cells at different stages.These findings provide a preliminary molecular mechanism explanation for the previous studies on the development and function of pulmonary lymphatic vessels.Moreover,our finding of c-Jun and Cdh13 regulated lymphatic function paves the way for future experiments to study pulmonary edema-related diseases such as transient tachypnea of the newborn and acute respiratory distress syndrome. |
