Transcriptome dynamics in developing larynx, trachea, and esophagus

The larynx, trachea, and esophagus share origin and proximity during embryonic development, with clinical and experimental evidence supporting the existence of neurophysiological, structural, and functional interdependencies before birth. This investigation provides the first comprehensive transcriptional profiling of all three organs during embryonic organogenesis, where differential gene expression gradually assembles the identity and complexity of these proximal organs from a shared origin in the anterior foregut. Through the application of bulk RNA sequencing and gene network analysis of differentially expressed genes (DEGs), both within and across developing embryonic mouse larynx, esophagus, and trachea, we identified co-expressed modules of genes enriched for key biological processes. Organ-specific temporal patterns of gene activity corresponding to gene modules within and across shared tissues during embryonic development (E10.5-E18.5) are described, and the laryngeal transcriptome during vocal fold development and maturation from birth to adult is characterized in the context of laryngeal organogenesis. The findings of this study provide new insights into interrelated gene sets governing organogenesis of this tripartite organ system within the aerodigestive tract, with relevance to multiple families of disorders defined by cardiocraniofacial syndromes. Wild type FVB/N mice males and females were mated, when vaginal plugs were found noon of that day was designated as embryonic day (E) 0.5. For RNA-Seq, pregnant females were sacrificed at E10.5, E11.5, E13.5, E15.5, E18.5, mouse larynges, trachea and esophagus were dissected, tissues were pooled together from 10 animals at E10.5, E11.5, E13.5, E18.5 time points, and 20 animals at the E15.5 timepoint. Approximately ~1-3 mm pieces of tissue were excised separately from the LPh/larynx (E10.5-Adult), esophagus (E10.5-E18.5) dorsoposterior to the larynx, and trachea (E10.5-E18.5) ventroposterior to the larynx with two medial biological replicates used at each timepoint. Tissue was collected anteriorly at the level of the LPh (E10.5 and E11.5) and vocal folds (E13.5 to adult) for the larynx and immediately posterior to the larynx in the case of both the esophagus and trachea. Additional timepoints for postnatal stage 0 (P0) and adult (6-8 weeks) larynx were included in the "within tissue" laryngeal analysis, where bulk tissue was collected at the level of the bilateral vocal folds. All tissues were micro-dissected in an RNase-free environment and immersed in RNAlater (Qiagen, Valencia, CA) at 4°C overnight, and then transferred to 80°C for long term storage prior to RNA extraction.喉、气管与食管在胚胎发育阶段共享起源且位置邻近，已有临床与实验证据证实，出生前三者之间存在神经生理、结构及功能层面的相互依存关系。 本研究首次对胚胎器官发生期这三种器官开展全面转录组分析——胚胎发育过程中，差异基因表达从前肠前部的共同起源位点出发，逐步赋予这些邻近器官各自的身份特征与复杂度。 通过对发育中的小鼠喉、食管、气管内部及跨器官的差异表达基因（Differentially Expressed Genes, DEGs）进行批量RNA测序与基因网络分析，本研究鉴定出了富集关键生物学过程的共表达基因模块。 本研究描述了胚胎发育阶段（E10.5至E18.5）中，共享组织内部及跨组织的基因模块对应的器官特异性基因表达时序模式，并在喉器官发生的背景下，解析了从出生到成年阶段声带发育与成熟过程中的喉转录组特征。 本研究结果为解析气消化道内这一三部分器官系统的器官发生相关的协同基因集提供了新视角，其研究发现与一类以心颅面综合征为特征的多种疾病家族均具有相关性。 将野生型FVB/N小鼠雌雄合笼交配，当日检测到阴道栓的正午时刻被定为胚胎第0.5天（E0.5）。 为开展RNA测序实验，分别在E10.5、E11.5、E13.5、E15.5及E18.5时间点处死孕鼠，解剖获取其喉、气管与食管；其中E10.5、E11.5、E13.5及E18.5时间点的组织混合自10只小鼠，E15.5时间点的组织混合自20只小鼠。 分别从喉咽部/喉（E10.5至成年）、喉背侧后方的食管（E10.5至E18.5）以及喉腹侧后方的气管（E10.5至E18.5）切取约1~3mm的组织块；每个时间点设置两份独立生物学重复。 喉组织的取材位置为：E10.5与E11.5阶段取材于喉咽部水平的前侧区域，E13.5至成年阶段取材于声带区域；而食管与气管组织均取材于喉的紧邻后侧区域。 在"组织内部"喉分析中，额外纳入了出生后第0天（P0）与成年（6~8周）阶段的喉组织样本，其取材位置为双侧声带区域的整块组织。 所有组织均在无RNA酶的环境下进行显微解剖，随后置于RNAlater（Qiagen, Valencia, CA）中4℃过夜保存，之后转移至-80℃长期储存，直至开展RNA提取。