Sp3 is essential for normal lung morphogenesis and cell cycle progression during mouse embryonic development. Sp3 is essential for normal lung morphogenesis and cell cycle progression during mouse embryonic development

Members of the Sp family of transcription factors regulate gene expression via binding GC boxes within promoter regions. Unlike Sp1, which stimulates transcription, the closely related Sp3 can either repress or activate gene expression and is required for perinatal survival in mice. Here we use RNAseq and cellular phenotyping to show how Sp3 regulates murine fetal cell differentiation and proliferation. Homozygous Sp3-/- mice were smaller than WT and Sp+/- littermates, died soon after birth, and had abnormal lung morphogenesis. RNAseq of Sp3-/- fetal lung mesenchymal cells identified alterations in extracellular matrix production, developmental signaling pathways, and myofibroblast/lipofibroblast differentiation. The lungs of Sp3-/- mice contained multiple structural defects, with abnormal endothelial cell morphology, lack of elastic fiber formation, and accumulation of lipid droplets within mesenchymal lipofibroblasts. Sp3-/- cells and mice also displayed cell cycle arrest, with accumulation in G0/G1 and reduced expression of numerous cell cycle regulators including Ccne1. These data detail the global impact of Sp3 on in vivo mouse gene expression and development. Development • Accepted manuscript lung mesenchymal cells identified alterations in extracellular matrix production, developmental signaling pathways, and myofibroblast/lipofibroblast differentiation. The lungs of Sp3-/- mice contained multiple structural defects, with abnormal endothelial cell morphology, lack of elastic fiber formation, and accumulation of lipid droplets within mesenchymal lipofibroblasts. Sp3-/- cells and mice also displayed cell cycle arrest, with accumulation in G0/G1 and reduced expression of numerous cell cycle regulators including Ccne1. These data detail the global impact of Sp3 on in vivo mouse gene expression and development. Overall design: Freshly isolated E15 fetal lung mesenchymal cells were isolated and passaged twice before being lysed in TRIzol for RNA isolation. RNA samples were then processed for RNAseq.

Sp家族转录因子（Sp family of transcription factors）通过结合启动子区域内的GC盒（GC boxes）调控基因表达。与可激活转录的Sp1不同，亲缘关系密切的Sp3既能够抑制也可以激活基因表达，且对小鼠的围产期存活不可或缺。本研究借助RNA测序（RNAseq）与细胞表型分析技术，阐明了Sp3对小鼠胎细胞分化与增殖的调控机制。纯合子Sp3敲除（Sp3-/-）小鼠的体型小于野生型（WT）及Sp3杂合（Sp+/-）同窝小鼠，出生后不久便死亡，且存在肺形态发生异常。对Sp3-/-胎肺间质细胞的RNAseq分析显示，其细胞外基质生成、发育信号通路以及肌成纤维细胞/脂成纤维细胞的分化均出现异常改变。Sp3-/-小鼠的肺部存在多处结构缺陷，包括内皮细胞形态异常、弹性纤维形成缺失，以及间质脂成纤维细胞内脂滴异常蓄积。Sp3-/-细胞与小鼠均表现出细胞周期阻滞现象，具体为G0/G1期细胞蓄积，且包括Ccne1在内的多种细胞周期调控因子的表达水平显著下调。本研究详细揭示了Sp3对小鼠体内基因表达与发育的全局调控效应。实验整体设计：将新鲜分离的E15胎肺间质细胞进行传代培养两次后，使用TRIzol裂解细胞以提取RNA，随后对RNA样本进行RNA测序建库分析。