Fig. 3. Auditory system development in Six1 homozygotes. (A,B) All Six1 homozygotes die at birth and exhibit severe auditory system defects involving the outer (arrow), middle and inner ears, as well as other defects. (C,D) Microdissected middle ear ossicles from E18.5 wild-type and Six1^-/- embryos. In the mutant, the incus (in) is malformed and fused with the malleus (ma) (arrowheads) and the stapes (st) is absent. The short process (sp) of the malleus is often missing (arrow) and the long process (lp) is also shortened. (E,F) Transverse sections of E10.5 Six1 heterozygous and homozygous embryos stained with Hematoxylin and Eosin showing the developing otic vesicle (ov) and the vestibuloacoustic ganglion (gVIII). In Six1^-/- embryos, although the otic vesicle formed, it appeared much smaller and abnormal (arrow) and the gVIII is absent (arrowhead). (G,H) Transverse sections of E12.5 wild-type and Six1 mutant embryos stained with X-gal for Six1^lacZ and counterstained with diluted Hematoxylin showing the developing inner ear, Six1^lacZ expression in the utricle and saccule region, semicircular canals, cranial ganglia gIX, gVIII and gV in the heterozygotes. However, in Six1^-/- embryos, only malformed semicircular canal-like structure was observed (arrow). Other inner ear structures are not formed and gIX (arrowhead) and gVIII (asterisk) are absent in the homozygotes. (I,J) TUNEL analysis of transverse sections through the ear region of Six1^+/- and Six1^-/- embryos at E9.5. Numerous apoptotic cells are only detected in the lateral wall of Six1^-/- otic vesicle (arrow). Scale bars: 100 µm.