Lin28 expression affect embryonic lung development

Lin28A and its paralog Lin28B are RNA binding proteins that regulate gene expression by inhibition of the maturation of the Per/Pri Let-7 miRNAs family and also via Let-7 independent mechanism. To study the effect of Lin28A ectopic-expression on mouse embryonic development we used a transgenic mice strain that combine a Cre-Lox system and a Tet-On system (herein Lox-stop-Lox-TetOn-Lin28A mice) and enable spatial and temporal control of transgenic Lin28A over-expression. Here we show that global Lin28A over-expression (by crossing Lox-stop-Lox-TetOn-Lin28A males with VasaCre females) in the mouse embryo prevents embryonic lung development. To study the precise developmental stage affected by Lin28A expression we preformed RNA sequencing analysis for transgenic and control lungs from E12.5, E14.5, E16.5 and E18.5 embryos. This analysis revealed that the development of the lung was delayed but not completely abrogated by Lin28A expression. We further showed that the effect of Lin28A expression in the mesenchymal cells (by crossing Lox-stop-Lox-TetOn-Lin28A males with Wt1Cre females) of the lung leads to the same phenotype as global Lin28A expression while expression in the lung epithelial cells (by crossing the Lox-stop-Lox-TetOn-Lin28A males with Nkx2.1Cre females) result in a milder developmental phenotype. RNA seq analysis was preformed for the following samples in triplicate (unless otherwise indicated): E12.5, E14.5, E16.5, E18.5 (5 repeats) control lungs. E12.5, E14.5 (4 repeats), E16.5, E18.5 (5 repeats) transgenic lungs derived from embryos with global Lin28A overexpression. E18.5 transgenic lungs derived from embryos with specific Lin28A expression in the lung epithelial cells. E18.5 transgenic lungs derived from embryos with Lin28A expression in the lung mesenchymal cells (duplicate).

Lin28A及其同源旁系蛋白Lin28B是一类RNA结合蛋白（RNA binding protein），可通过抑制Per/Pri Let-7微RNA家族的成熟，以及不依赖于Let-7的通路调控基因表达。为研究Lin28A异位表达对小鼠胚胎发育的影响，本研究采用结合了Cre-Lox重组系统（Cre-Lox system）与Tet-On诱导系统（Tet-On system）的转基因小鼠品系（本文中称为Lox-stop-Lox-TetOn-Lin28A小鼠），可实现转基因Lin28A过表达的时空精准调控。本研究通过将Lox-stop-Lox-TetOn-Lin28A雄性小鼠与VasaCre雌性小鼠杂交，使小鼠胚胎实现全局性Lin28A过表达，结果显示该操作会阻断胚胎肺发育。为明确Lin28A表达影响的具体发育阶段，我们对E12.5、E14.5、E16.5及E18.5胚胎的转基因与对照肺组织进行了RNA测序（RNA sequencing）分析。该分析结果显示，Lin28A表达会导致肺发育延迟，但并未完全阻断肺发育。我们进一步证实，通过将Lox-stop-Lox-TetOn-Lin28A雄性小鼠与Wt1Cre雌性小鼠杂交，在肺间质细胞中特异性表达Lin28A，所产生的表型与全局性Lin28A过表达一致；而通过与Nkx2.1Cre雌性小鼠杂交，在肺上皮细胞中表达Lin28A，则仅会引发程度更温和的发育表型。本研究对以下样本进行了RNA测序分析，默认每个样本设置三次生物学重复（另有说明除外）：E12.5、E14.5、E16.5及E18.5的对照肺组织（各5次生物学重复）；全局性Lin28A过表达胚胎的转基因肺组织：E12.5、E16.5（各5次重复），E14.5（4次重复），E18.5（5次重复）；肺上皮细胞特异性Lin28A表达胚胎的E18.5转基因肺组织；肺间质细胞特异性Lin28A表达胚胎的E18.5转基因肺组织（2次重复）。