Figure 6. Activin signaling is required for patterning and maturation of hair cells.

(A–B) FST overexpression at E12.5 delays hair cell maturation and causes a mild overproduction of inner hair cells. FST transgenic (R26-FST) embryos and their control (single transgenic) littermates were exposed to dox starting at E12.5 until tissue harvest at E17.5. (A) Shown are the apical surfaces of hair cells located in the base, mid and apex of control and R26-FST transgenic cochlear sensory epithelia. Phalloidin labels actin-rich stereocilia of inner (white arrowhead) and outer hair cells (white bar). Red asterisks mark ectopic inner hair cells. White arrows mark location of missing outer hair cells. Scale bar 50 µm. (B) Graphed are the number of ectopic inner hair cells (IHC) within the most basal segment (0–0.5 mm) and within the entire length (0–4 mm) of control (Ctrl, gray) and FST overexpressing (FST, purple) cochleae. Data expressed as mean ± SEM (n = 3 animals per group, ****p<0.0001, student’s t-test). (C-D) Conditional ablation of the Inhba gene delays hair cell maturation and causes a mild overproduction of inner hair cells. (C) Shown are the apical surfaces of hair cells located in the base, mid and apex of stage P0 Inhba fl/fl (control) and Pax2-Cre Inhba fl/fl (Inhba cKO) cochlear sensory epithelia. Phalloidin labels actin-rich stereocilia of inner (white arrowhead) and outer hair cells (white bar). Red asterisks mark ectopic inner hair cells. Scale bar 50 µm. (D) Graphed are the number of ectopic inner hair cells (IHC) within the most basal segment (0–0.5 mm) and within the entire length (0–4 mm) of control (Ctrl, gray) and Inhba mutant (Inhba cKO, orange) cochleae. Data expressed as mean ± SEM (n = 3 animals per group, *p≤0.05, ***p<0.001).

Figure 6—figure supplement 1. Characterization of Activin-type ligand expression in cochlear epithelial cells.

(A) Relative mRNA expression levels of Activin–type ligands (Inhba, Inhbb, Gdf11, Mstn) and Activin antagonist Inhibin A (Inha) in cochlear epithelia cells at the onset of (E13.5), during (E14.5, E15.5), and after cochlear differentiation (E18.5). Atoh1 mRNA expression is a marker of nascent hair cells. Data are mean ± SEM (n = 3 technical replicates). B: Plotted is the difference in cycle threshold (ΔCT) between target genes and the reference gene Rpl19 in the analysis shown in A. Note that a ΔCT level of 14 or higher indicates that a gene is expressed at a very low level close to the limit of detection. Data are mean ± SEM (n = 3 technical replicates).