Bulk RNA-Seq of Foxe1KO and control Nkx2-1/mKO2+ mouse ESC-derived cells. Bulk RNA-Seq of Foxe1KO and control Nkx2-1/mKO2+ mouse ESC-derived cells

Functional thyroid follicles generation in vitro is obtained at high efficiency by doxycycline-mediated expression of Nkx2-1 and Pax8, two proteins involved in early specification of thyroid lineage. Although those factors are sufficient to induce thyroid specification in vitro, other players, such as Foxe1, are also present in early thyroid primordium and its loss-of-function is responsible for thyroid congenital defects in mice and humans. Here we show that Foxe1KO mESCs can be induced to differentiate towards thyroid lineage but the efficiency of thyroid follicular cells generated is drastically low. Moreover, correct expression of maturation genes and capacity to produce thyroid hormone in vitro are completely disrupted. On the other hand, Nkx2-1 expressing cells derived from Foxe1KO have an unexpected ability to deviate to a lung epithelium differentiation program and form organoids containing multiple lung cell types. These findings demonstrate that Foxe1 is required to reinforce thyroid cell fate in vitro and to promote terminal maturation of thyroid follicles. Overall design: Foxe1KO and control cells from Nkx2-1 reporter line were cultured following the differentiation protocol. 10000 Nkx2-1+ (mKO2+) cells per condition were sorted (FACS Aria; BD Bioscience) at day10 and day22 time points of the differentiation protocol. Cells were collected directly into Qiazol lysis reagent (Qiagen) and RNA isolation performed with miRNeasy micro kit (Qiagen) following manufacturer’s indications.Ovarion Solo RNA-seq Systems (NuGen) was employed, as indicated by the manufacturer. Multiplexed libraries (10ρM) were loaded onto flow cells and sequenced on the HiSeq 1500 system (Illumina) in high-output mode using the HiSeq Cluster Kit v4 (Illumina).

通过强力霉素（doxycycline）介导的Nkx2-1与Pax8表达，可高效体外获得功能性甲状腺滤泡。Nkx2-1与Pax8是参与甲状腺谱系早期特化的两种核心蛋白。尽管上述两种因子足以在体外诱导甲状腺谱系特化，但早期甲状腺原基中还存在包括叉头框E1（Foxe1）在内的其他调控因子；该因子功能缺失会导致小鼠与人类的甲状腺先天性发育缺陷。
本研究显示，Foxe1基因敲除（Foxe1KO）的小鼠胚胎干细胞（mouse embryonic stem cells, mESCs）可被诱导向甲状腺谱系分化，但所得甲状腺滤泡细胞的生成效率显著降低。此外，体外环境下甲状腺成熟相关基因的正常表达以及甲状腺激素合成能力完全丧失。另一方面，源自Foxe1KO细胞的表达Nkx2-1的细胞，会意外偏离预设的甲状腺分化程序，转向肺上皮细胞分化，并形成包含多种肺细胞类型的类器官。
上述研究结果表明，Foxe1在体外可强化甲状腺细胞命运，并促进甲状腺滤泡的终末成熟。
实验设计：将来自Nkx2-1报告基因细胞系的Foxe1KO细胞与对照细胞，按照既定分化方案进行培养。在分化方案的第10天与第22天，通过流式细胞分选仪（FACS Aria；BD Bioscience）对每个实验组的10000个Nkx2-1+（mKO2+）细胞进行分选。将收集的细胞直接加入Qiazol裂解试剂（Qiagen），并按照制造商说明书使用miRNeasy微量试剂盒（Qiagen）进行RNA提取。采用Ovarion Solo RNA-seq系统（NuGen）进行文库制备，操作遵循制造商指南。将浓度为10ρM的多重测序文库加载至流动槽，使用HiSeq簇生成试剂盒v4（Illumina），在HiSeq 1500测序系统（Illumina）上以高产出模式进行测序。