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. 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).