Figure S5.

3D organization and ontogenesis of the ear, related to Figure 2

(A–J) 3D LSFM image of the inner and middle ear in a PCW7.5 human embryo stained with MHC, Sox9 and Synaptophysin, cleared and segmented with syGlass. (A) is a merge of all channels, with segmented elements pseudocolored. (B)–(G) shows the two muscles and the three bones of the middle ear, after segmentation (B, C, E, and F) and 3D surface rendering (D and G). (H) is a 3D rendering view showing all elements together: the petrous bone template (gray), the stapedius (light blue) connected to the stapes, the tensor tympani connected to the malleus, the facial nerve (VII), and its chorda tympani (CT) branch. (I) LSFM image showing the relative positions of the two muscles, together with the cochlea (Co), facial nerve (VII), chorda tympani (CT), cochlear nerve (CN), and vestibulocochlear/auditory nerve (VIII). (J) 3D rendering of the cochlea (Co), saccule (S), utricule (U), anterior (A), posterior (P), and lateral (L) semicircular canals together with the vestibular (VN) and cochlear (CN) nerves. The cochlea and canals were segmented based on the background of the Sox9 staining.

(K–M) 3D ontogenesis of external ear muscles in human embryo (K) and fetuses (L and M), immunostained with anti-MHC antibodies (K–M) and ChAT (L). The overlays are surface shading images of the auricle/pinna (gray). Muscles have been segmented using VR and pseudocolored. The color code for muscle identification is on the upper right inset. (K) shows that all individual muscles have started to emerge at PCW7.5. The arrow indicates the developing external auditory meatus. (L) All muscles are innervated by ChAT+ motor nerves (arrowheads show innervating branches). (M) shows the muscles in a PCW11.9 fetus. The size of the muscles has increased, and they now form a ring around the ear.

Scale bars: 500 μm in (A), (B), and (H)–(J); 300 μm in (C) and (E)–(G); 200 μm in (D); and 500 μm in (K)–(M).