Analysis of FGF20-regulated genes in differentiating cochlear hair and supporting cell progenitors via ribosome affinity purification

Background: Understanding the developmental mechanisms that regulate hair cell differentiation in the cochlea is essential to designing genetic therapies for acquired hearing loss due to hair cell loss or damage. We have previously identified Fibroblast Growth Factor 20 (FGF20) as having a key role in hair cell and supporting cell differentiation and patterning in the mouse cochlear sensory epithelium. To investigate the genetic landscape regulated by FGF20 signaling in hair cell and supporting cell progenitors, we employ Translating Ribosome Affinity Purification (TRAP) combined with Next Generation mRNA Sequencing (TRAPseq). Methods: In mice, we used Fgf20-Cre to activate ROSA-fsTRAP in hair cell and supporting cell progenitors, and then collected translating mRNA using TRAP from Fgf20+/- (control) and Fgf20-/- cochleae. Library preparation and sequencing were done in two separate experiments, each with 12 samples that included 2 pre-TRAP (pre-immunoprecipitation) Fgf20+/- samples, 2 TRAP Fgf20+/- samples, 4 pre-TRAP Fgf20-/- samples, and 4 TRAP Fgf20-/- samples. Samples were sequenced via Illumina HiSeq 3000. We compared pre-TRAP (pre-immunoprecipitation) samples with TRAP mRNA samples to validate the TRAP technique as well as identify genes enriched in our target cell population. We also compared Fgf20+/- and Fgf20-/- TRAP mRNA samples to identify differentially expressed genes downstream of FGF20 during hair cell and supporting cell differentiation. Results: TRAPseq targeting the prosensory cell population effectively enriches for translating mRNA within this rare cell population. TRAPseq comparing Fgf20+/- and Fgf20-/- samples identified differentially expressed genes downstream of FGF20. These included FGF-response genes Etv4, Etv5, Etv1, and Dusp6, as well as genes associated with cochlea development and hearing, such as Hey1, Hey2, Heyl, Tectb, Fat3, Cpxm2, Sall1, and cell cycle regulators such as Cdc20. E14.5 cochlear mRNA collected with translating ribosome affinity purification from Fgf20+/- and Fgf20-/- mouse cochleae. Expression of L10a-eGFP protein was targeted to the Fgf20-Cre lineage. Each sample is pooled from 3-7 embryos of the same genotype.研究背景：解析耳蜗中调控毛细胞分化的发育机制，对于开发因毛细胞丢失或损伤所致获得性听力损失的基因治疗方案至关重要。我们此前已证实成纤维细胞生长因子20（Fibroblast Growth Factor 20, FGF20）在小鼠耳蜗感觉上皮的毛细胞与支持细胞分化及模式形成中发挥关键作用。为探究FGF20信号通路在毛细胞及支持细胞祖细胞中调控的遗传调控网络，本研究采用翻译核糖体亲和纯化（Translating Ribosome Affinity Purification, TRAP）结合下一代mRNA测序（Next Generation mRNA Sequencing, TRAPseq）的实验策略。 实验方法：在小鼠模型中，我们利用Fgf20-Cre启动ROSA-fsTRAP在毛细胞与支持细胞祖细胞中的表达，随后分别从Fgf20+/-（对照组）与Fgf20-/-小鼠的耳蜗中通过TRAP技术分离翻译中的mRNA。文库构建与测序分两次独立实验完成，每次实验包含12个样本：2份pre-TRAP（免疫沉淀前）Fgf20+/-样本、2份TRAP Fgf20+/-样本、4份pre-TRAP Fgf20-/-样本以及4份TRAP Fgf20-/-样本。所有样本均通过Illumina HiSeq 3000平台完成测序。我们通过对比pre-TRAP与TRAP mRNA样本，验证TRAP技术的有效性，并筛选目标细胞群体中富集的基因；同时对比Fgf20+/-与Fgf20-/-的TRAP mRNA样本，鉴定毛细胞与支持细胞分化过程中FGF20下游的差异表达基因。 实验结果：靶向前感觉细胞群体的TRAPseq可有效富集该稀有细胞群体中的翻译mRNA。对比Fgf20+/-与Fgf20-/-样本的TRAPseq分析，成功鉴定出FGF20下游的差异表达基因，包括FGF应答基因Etv4、Etv5、Etv1及Dusp6，同时还包含与耳蜗发育及听觉功能相关的基因如Hey1、Hey2、Heyl、Tectb、Fat3、Cpxm2、Sall1，以及细胞周期调控因子如Cdc20。本数据集获取自E14.5天Fgf20+/-与Fgf20-/-小鼠耳蜗中通过翻译核糖体亲和纯化得到的mRNA，其中L10a-eGFP蛋白的表达靶向Fgf20-Cre标记的细胞谱系。所有样本均由3-7只相同基因型的胚胎组织混合制备。