Transcriptomic analysis of P0 mouse lung lymphatic endothelial cells

Fluid clearance mediated by lymphatic vessels is known to be essential for lung inflation and gas exchange function during the transition from prenatal to postnatal life, yet the molecular mechanisms that regulate lymphatic function remain unclear. Here, we profiled the molecular features of lymphatic endothelial cells (LECs) in embryonic and postnatal day (P) 0 lungs by single-cell RNA-seq analysis. We identified that the expression of c-JUN is transiently upregulated in P0 LECs. Conditional knockout of Jun in LECs impairs the opening of lung lymphatic vessels at birth, leading to fluid retention in the lungs and neonatal death. We further demonstrated that increased mechanical pressure induces the expression of c-JUN in LECs. c-JUN regulates the opening of lymphatic vessels by modulating remodeling of the actin cytoskeleton in LECs. Our study established the essential regulatory function of c-JUN-mediated transcriptional responses in facilitating lung lymphatic fluid clearance at birth. Control and Jun knockout LECs from Prox1-GFP mouse lungs were isolated at P0 by FACS. LECs isolated from three replicates of each group were identically mixed and then processed to RNA amplification following the protocol of Arcturus RiboAmp HS PLUS RNA amplification kit before RNA sequencing. Datasets were generated named as "Jun-Ctrl", and "Jun-cKO".

已知在从产前至产后的生命过渡阶段，淋巴管介导的液体清除对肺扩张与气体交换功能至关重要，但调控淋巴管功能的分子机制仍未明确。本研究通过单细胞RNA测序（single-cell RNA-seq）分析，对胚胎期及产后0天（P0）肺组织内的淋巴管内皮细胞（lymphatic endothelial cells, LECs）的分子特征进行了表征。本研究发现c-JUN的表达在P0期LECs中呈瞬时上调状态。在LECs中条件性敲除Jun基因会损伤出生时肺淋巴管的开放功能，进而引发肺内液体潴留与新生小鼠死亡。本研究进一步证实，机械压力升高可诱导LECs中c-JUN的表达。c-JUN通过调控LECs内的肌动蛋白细胞骨架重塑，实现对淋巴管开放功能的调节。本研究明确了c-JUN介导的转录应答在出生时促进肺淋巴管液体清除过程中的关键调控作用。本研究从P0天Prox1-GFP转基因小鼠的肺组织中，通过荧光激活细胞分选（fluorescence-activated cell sorting, FACS）分离得到对照组与Jun敲除组的LECs。每组取3次生物学重复分离得到的LECs进行等量混合，随后按照Arcturus RiboAmp HS PLUS RNA扩增试剂盒的操作流程进行RNA扩增，再进行RNA测序。最终生成的测序数据集分别命名为"Jun-Ctrl"与"Jun-cKO"。