Transcriptome Dynamics in the Developing Larynx, Trachea, and Esophagus

Abstract

The larynx, trachea, and esophagus share origin and proximity during embryonic development. Clinical and experimental evidence support the existence of neurophysiological, structural, and functional interdependencies before birth. This investigation provides the first comprehensive transcriptional profile 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. By applying bulk RNA sequencing and gene network analysis of differentially expressed genes (DEGs) 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 adulthood is characterized in the context of laryngeal organogenesis. The findings of this study provide new insights into interrelated gene sets governing the organogenesis of this tripartite organ system within the aerodigestive tract. They are relevant to multiple families of disorders defined by cardiocraniofacial syndromes.

Keywords: RNAseq; development; esophagus; larynx; morphogenesis; trachea.

FIGURE 1

Global transcriptome structure across the esophagus, trachea, and larynx during embryonic development, (A) Schematic of developing embryos shows the color key for organ identity and developmental stage across the study timespan above hematoxylin and eosin histological sections at the level of the developing vocal folds during each time point. (B) PCA analysis demonstrates 2D representation of whole-tissue transcriptome principal components at E10.5, E11.5, E13.5, E15.5, and E18.5 across embryonic development of the Lr, larynx; Es, esophagus; Tr, trachea; (C) Heatmap of differentially expressed genes (DEGs) (n = 3,472) constructed from normalized expected counts across embryonic development of the primitive laryngopharynx (LPh), larynx (Lr), esophagus (Es), and trachea (Tr) at E10.5, E11.5, E13.5, E15.5, and E18.5 with expression measures averaged across replicates within each tissue/age combination and then converted within each gene into a z-score for improved visual comparison.

FIGURE 2

Hierarchal clustering of gene modules within developing larynx, esophagus, and trachea, (A) Heatmap of differentially expressed genes (DEGs) (n = 4,519) constructed from normalized expected counts during embryonic development of primitive laryngopharynx/larynx (LPh/Lr) across E10.5, E11.5, E13.5, E15.5, E18.5, P0, and Adult (6–8 weeks) with expression measures averaged across replicates within each tissue/age combination and then converted within each gene into a z-score for improved visual comparison; (B) PCA analysis demonstrates 2D representation of whole-tissue transcriptome principal components at each timepoint for the LPh/Lr; (C) Heatmap of DEGs in the esophagus (Es) (n = 2,766) across E10.5, E11.5, E13.5, E15.5, and E18.5; (D) PCA analysis demonstrates 2D representation of whole-tissue transcriptome principal components at each timepoint for the Es; (E) Heatmap of DEGs in the trachea (Tr) (n = 2,921) constructed from normalized expected counts during embryonic development of trachea across E10.5, E11.5, E13.5, E15.5, and E18.5; (F) PCA analysis demonstrates 2D representation of whole-tissue transcriptome principal components at each timepoint for the Tr.