RNAseq profiling of multiciliated cells. Xenopus laevis

To determine what genes are upregulated in multiciliated cells, we manipulated Xenopus laevis ectoderm to either make more or fewer of this cell type with multiple approaches across multiple timepoints. By finding differentially expressed genes in common across all comparisons in which multiciliated cell number changed, we obtained a robust, but conservative, core group of multiciliated cell genes. Overall design: We suppressed multiciliated cell development by activating the notch pathway with an injected mRNA encoding the intracellular domain of notch (icd) or by injecting an mRNA encoding a dominant-negative form of multicilin (dnmcidas). Conversely, we promoted multiciliated cell differentiation by blocking notch signaling with a DNA-binding mutant of Suppressor of Hairless (dbm), or by overexpressing an inducible form of multicilin (mcidas). We also coinjected these constructs in a way aimed at causing the greatest change in multiciliated cells and reducing background transcriptional programs not associated with these cells: for example, we coinjected icd with mcidas, in order to reduce other cell types specified by notch. After injecting, we isolated ectoderm surgically and, when injected with multicilin, induced at mid-stage 11. We then harvested RNA at 3, 6, and 9 hours after induction, roughly corresponding to stages 13, 16, and 18 and performed poly-a+ RNAseq (Illumina Truseq v2). We then aligned reads to X. laevis gene models (Mayball version, Chung and Kwon et al. 2014) and took the intersection of genes differentially expressed between all comparisons in which the multiciliated cell number dramatically changed (icd vs. icd + mcidas, icd vs. dbm, dbm vs. dbm + dnmcidas) to obtain a core list of multiciliated cell genes.为鉴定多纤毛细胞（multiciliated cell）中上调的基因，我们采用多种方法、在多个时间点对非洲爪蟾（Xenopus laevis）外胚层进行干预，以调控该细胞类型的数量增减。通过在所有多纤毛细胞数量发生变化的比对组合中筛选共有差异表达基因，我们获得了一组稳健且保守的多纤毛细胞核心基因集。 整体实验设计：我们通过两种方式抑制多纤毛细胞发育：一是注射编码Notch胞内结构域（intracellular domain of notch, icd）的mRNA以激活Notch信号通路，二是注射编码显性负效型多纤毛素（multicilin, dnmcidas）的mRNA。反之，我们通过两种手段促进多纤毛细胞分化：一是采用毛发抑制因子（Suppressor of Hairless）的DNA结合突变体（dbm）阻断Notch信号通路，二是过表达可诱导型多纤毛素（multicilin, mcidas）。我们还采用共注射构建体的策略，以最大化改变多纤毛细胞数量并减少与该细胞类型无关的背景转录程序：例如，我们共注射icd与mcidas，以减少Notch信号通路调控的其他细胞类型。 注射完成后，我们通过手术分离外胚层；对于注射了可诱导型多纤毛素的样本，我们在胚胎发育中期11期时启动其诱导表达。随后分别在诱导后的3、6、9小时（大致对应胚胎发育13、16、18期）收集样本并提取RNA，随后进行polyA+ RNA测序（采用Illumina Truseq v2建库方案）。 随后，我们将测序读段比对至非洲爪蟾（Xenopus laevis）基因注释模型（Mayball版本，Chung与Kwon等，2014），并取所有多纤毛细胞数量发生显著变化的比对组合（icd vs. icd+mcidas、icd vs. dbm、dbm vs. dbm+dnmcidas）之间的差异表达基因的交集，最终得到多纤毛细胞核心基因列表。