Mechanisms of larynx and vocal fold development and pathogenesis

Abstract

The larynx and vocal folds sit at the crossroad between digestive and respiratory tracts and fulfill multiple functions related to breathing, protection and phonation. They develop at the head and trunk interface through a sequence of morphogenetic events that require precise temporo-spatial coordination. We are beginning to understand some of the molecular and cellular mechanisms that underlie critical processes such as specification of the laryngeal field, epithelial lamina formation and recanalization as well as the development and differentiation of mesenchymal cell populations. Nevertheless, many gaps remain in our knowledge, the filling of which is essential for understanding congenital laryngeal disorders and the evaluation and treatment approaches in human patients. This review highlights recent advances in our understanding of the laryngeal embryogenesis. Proposed genes and signaling pathways that are critical for the laryngeal development have a potential to be harnessed in the field of regenerative medicine.

Keywords: Congenital; Embryology; Laryngeal; Vocal cords.

Fig. 1

Morphology of the murine larynx and vocal folds. a–f Schematic illustrations of the murine laryngeal cartilages and muscles. Sagittal section through the murine larynx showing the position of the VF (in yellow) and epiglottis, thyroid cartilage, alar cartilage, arytenoid cartilages and cricoid cartilage (a). Frontal view of the murine larynx showing the attachment of the cricothyroid muscle to the thyroid and cricoid cartilages (b). Sagittal section through the murine larynx showing the attachment of the thyroarytenoid muscle, lateral cricoarytenoid muscle, superior and posterior cricoarytenoid muscles (c). Transverse sections of the murine VF, the cranial (anterior) section (d) and caudal (posterior) section (e). Bracketed region in the panel of e shows the detailed morphology of the right vocal fold (f). g, h Hematoxylin–eosin staining showing morphology of the murine vocal folds in coronal (g) and transverse section (h). Bracketed regions in the panel of g, h show detailed morphology of the right vocal fold in coronal and transversal sections, respectively. Scale bar 500 µm. A anterior, Ar alar cartilage, AC arytenoid cartilage, CC cricoid cartilage, CT cricothyroid muscle, D dorsal, Ep epithelium, Epi epiglottis, LP lamina propria, P posterior, PCA posterior cricoarytenoid muscle, SCA superior cricoarytenoid muscle, TA thyroarytenoid muscle, TC thyroid cartilage, V ventral, VF vocal fold

Fig. 2

Developmental origin of laryngeal and vocal fold structures. a Schematic illustration of major cell populations that contribute to the laryngeal and vocal fold structures including anterior foregut endoderm (in blue), lateral mesoderm (in red) and neural crest cells (in yellow). b Schematic illustration showing that anterior foregut endoderm-derived cells expressing ShhCre^+/−TdTom Red give rise primarily to the laryngeal and VF epithelium (in blue). c Schematic illustration showing that cells of the lateral mesoderm-derived cells expressing Mesp1Cre^+/−TdTom Red give rise to the cricoid cartilage, arytenoid cartilages, intrinsic laryngeal muscles, and the intermediate lamina of the thyroid cartilage in the caudal-most region of the thyroid cartilage (in red). d Schematic illustration showing that neural crest-derived cells expressing Wnt1Cre^+/−mGFP contribute to formation of the loose connective tissue in the ventral lamina propria, nervous tissue and thyroid cartilage, except for its medial caudal-most portion (in yellow). AC arytenoid cartilage, AFE anterior foregut endoderm, CC cricoid cartilage, D dorsal, EP vocal fold epithelium, LM lateral mesoderm, NCC neural crest cell, TA thyroarytenoid muscle, TC thyroid cartilage, V ventral

Fig. 3

Schematic illustration of larynx and vocal fold development. a, b Time scales of the laryngeal and VF development showing that the human larynx and VF (a) pass through similar developmental stages as the murine larynx and VF (b). These stages include the specification of the laryngeal field, apposition of the lateral walls, EL formation, EL recanalization and maturation of the VF epithelium and mesenchymal structures during the fetal period (in humans) and postnatally (in mice). Schematic illustrations demonstrate the murine larynx and VF development in more detail. The first image shows the position of the laryngotracheal groove at the level of the fourth pharyngeal pouch at stage E 9.5. At E10.5, the primitive LPh initiates its formation. It becomes bilaterally compressed due to the approximation of the lateral walls to the center of the lumen. At E11.5, the closely juxtaposed lateral walls fuse and create the EL. Differentiation of mesenchymal structures initiates. At E13.5, the tracheoesophageal septum is formed and the EL begins its recanalization, due to the expansion of the laryngeal cecum and pharyngoglottic duct that exert pressure on the epithelial walls. During EL recanalization, laryngeal cartilages and muscles continue to differentiate. At E18.5, the VF are separated and mesenchymal structures are fully developed to anchor the growing VF. EL epithelial lamina, Es esophagus, LB lung bud, LC laryngeal cecum, LG laryngotracheal groove, LPh primitive laryngopharynx, PA pharyngeal arch, PD pharyngoglottic duct, PP pharyngeal pouch, RD respiratory diverticulum, Tr trachea, VF vocal fold

Fig. 4

Genes and signaling molecules participating in larynx and vocal fold development. a Genes and signaling molecules that control specification of the laryngeal field and early stages of primitive LPh remodeling. Shh is expressed in the LPh endoderm (red color) and likely controls the juxtaposition of Nkx2-1 (green solid line) and Sox2 (yellow solid line) expression domains in the LPh endoderm and cell proliferation. In the lamina propria, Gli genes along with genes involved in the pharynx segmentation, such as RA, Tbx1, β-Cat, Hox genes, Fgf signaling, contribute to initial differentiation of the mesenchymal cells and incorporation of migratory cell populations. (B) Genes and signaling molecules that control the fusion of lateral walls and simultaneous differentiation of supportive mesenchymal structures—cartilages (dark gray) and muscles (brown dashed lines). β-Catenin (blue color) expressed in the EL likely controls cell proliferation via Cyclin D1 expression and simultaneous differentiation of basal p63+ K8+ progenitors. In the lamina propria, laryngeal cartilages initiate differentiation into Sox9+ chondrocytes and MF-20+ myoblasts. Other genes and signaling molecules acting on differentiation of laryngeal cartilages and muscles include R-spondin2, Eph/EphrinB2 signaling. c Genes and signaling molecules that control EL recanalization and further differentiation of laryngeal cartilages (dark gray), muscle (brown dashed lines) and nerves (purple curvy lines). Proper stratification of VF basal progenitors has been linked to β-Catenin (blue color) which controls conversion of p63+ cytokeratin (K) 8+ basal progenitors into functional basal p63+, K8− cells capable of stratification. AC arytenoid cartilage, CC cricoid cartilage, EL epithelial lamina, LPh primitive laryngopharynx, TC thyroid cartilage