Fig. 4. Effects of ablating ventral midline structures on inner ear development. Chicken embryos are operated in ovo at E1.5, and further incubated and harvested at E2.5 for the assessment of Shh expression using whole-mount in situ hybridization (A,D,G,J,M) or at E7 for anatomical analysis of the inner ear using the paint-fill technique (C,F,I,L,O). Representative histological sections from ablated regions are also shown (B,E,H,K,N). Whole-mount (A) and section (B) showing Shh expression in the floor plate (FP) and notochord (noto) of control embryos. (C) Normal inner ear of controls at E7. (D-F) Both the notochord and ventral neural tube, including the floor plate are ablated in the region between R4-R7. (D,E) Floor plate (arrow) and notochord (arrowheads) associated Shh expression are missing in the ablated region. (F) The lack of ventral inner ear structures in an ablated embryo (asterisk). (G-I) Only the notochord underneath R4-R7 is ablated. (G,H) Neural tube-associated (arrow) but not notochord-associated (arrowheads) Shh expression is present in the ablated region. (I) The inner ear morphology of a notochord-ablated embryo is normal. (J-L) A region of the neural tube between R4-R7 is ablated. (J,K) Neural tube-associated Shh expression is missing in the ablated region (arrow), whereas the notochord-associated Shh expression is not disrupted (arrowheads). (L) Dorsal inner ear structures (asterisk) are missing after neural tube removal. (M-O) Both the notochord and neural tube are ablated. (M,N) Shh expression is absent from the ablated region. (O) A rudimentary inner ear results when both the notochord and neural tube are removed. Black asterisks indicate the positions of otocysts. Scale bars: in A, 100 µm for A,D,G,J,M; in B, 100 µm for B,E,H,K,N; in C, 500 µm for C,F,I,L,O.