Loss of RANK signaling leads to stem cell activation in post pregnancy mammary glands

The purpose of this study was to elucidate the molecular changes of the mammary epithelium induced by luminal cell specific RANK deletion. We have characterized the phenotypic consequences of RANK deletion in the luminal lineage of the mammary epithelium, and uncovered that Rank KO luminal cells have reduced long term renewal following sucessive pregnancies. Lineage tracing analysis has demonstrated that during pregnancy 2, Rank deleted luminal cells are repleace by a wt luminal cell population, derived from the basal epithelium. We therefore decided to perform RNA seq in resting mammary glands from virgin and parous (following pregnancy one) females, cells were FACS sorted based on the traditional markers CD24 and CD49f and in the case of luminal cells GFP (recombined cells). We have isolated luminal cells to identify the molecular pathways involved in reduced long term renewal oberved in the parous context; basal cells were isolated to adress the putative mechanims regulated the bipotency observed during second pregnancy Overall design: Animal were treated with doxicyclin (1mg/ml in the drinking water) between 3 and 8 weeks of age to induce cre recombination

本研究旨在阐明腔上皮细胞（luminal cell）特异性RANK缺失所诱导的乳腺上皮（mammary epithelium）的分子变化。我们已对乳腺上皮腔系中RANK缺失的表型后果进行了系统表征，并发现Rank敲除（Rank knockout, Rank KO）的腔上皮细胞在连续妊娠后长期自我更新能力显著降低。谱系示踪分析（lineage tracing analysis）表明，在第二次妊娠期间，Rank缺失的腔上皮细胞会被源自基底上皮的野生型（wild type, wt）腔上皮细胞群体所替代。因此，我们对未妊娠雌性小鼠与经产（经历过一次妊娠）雌性小鼠的静息乳腺组织开展RNA测序（RNA-seq）。实验中，细胞通过荧光激活细胞分选（fluorescence-activated cell sorting, FACS），以传统标志物CD24和CD49f作为分选依据，针对腔上皮细胞还额外结合GFP（重组细胞）特征进行分选。我们分离腔上皮细胞以鉴定经产环境中观察到的长期自我更新能力降低所涉及的分子通路；同时分离基底上皮细胞，以探究第二次妊娠期间观察到的双潜能性的潜在调控机制。实验设计：在小鼠3至8周龄期间，给予其饮用含有1mg/ml多西环素（doxycycline）的饮水，以诱导Cre重组酶（Cre recombinase）重组。