Tshz3 inactivation impairs the development of the nucleus ambiguus. A–B ′, Transverse sections of the medulla stained with cresyl violet from WT ( A , A ′) and Tshz3^lacZ/lacZ embryos ( B , B ′) at E18.5. Compact formation of the nA is not visible in Tshz3 mutants. C–H , Periferin in situ hybridization on transverse sections of E18.5 WT ( C–E ) and Tshz3^lacZ/lacZ ( F–H ). In Tshz3 mutants, the nA is strongly reduced in size ( C , F ). The facial nucleus ( D , G ), the nucleus dmnX, and the hypoglossal nucleus (XII) ( E , H ) appear normal in size. I , J , PHOX2b immunodetection on parasagittal sections through E15.5 hindbrain of WT ( I ) and Tshz3^lacZ/lacZ ( J ) embryos; rostral is left. Note the drastic reduction of the number of PHOX2b+ cells in the nA (indicated with a dotted line) of the mutant. K , L , Photomicrographs of parasagittal sections through the nA of E14.5 WT and Tshz3 mutant analyzed by TUNEL (red) and PHOX2b immunostaining (green). M , N , Immunostaining for PHOX2b (green) and activated caspase-3 (red) on sagittal sections of the nA from E14.5 WT ( M ) and Tshz3^lacZ/lacZ ( N ) embryos. Scale bars: A , B , 500 μm; C , D , F , G , 100 μm; E , H–J , 200 μm.