Fig. 5. Specification of facial branchio-motoneurons is not affected by Nkx6.1 inactivation. In situ hybridization with Isl1 (A,B) and Isl2 (D,E) on flat-mounted hindbrains of E10.5 wild-type (A,D) and Nkx6.1 mutant (B,E) embryos. (B) Nkx6.1 mutant embryos generate Isl1-positive cells throughout the hindbrain. (C) Quantification of Isl1-positive facial branchio-motoneurons at r4 levels of wild-type and Nkx6.1 mutant embryos at E10.5 and E12.5. Using immunohistochemistry with an anti-Isl1 antibody on coronal hindbrain sections, Isl1-positive nuclei on 12 representative sections for each genotype and age were counted. Values are shown as % of wild type, mean±s.d. (D,E) Isl2 marks somatic motoneurons and the otic ganglion. Isl2-positive motoneurons are not detected in hindbrains of Nkx6.1 mutants (E). (F-S) In situ hybridization with Olig2 (F,G), Irx3 (H,I), Dbx2 (J,K), Dbx1 (L,M) and Ebf1 (R,S) and co-immunofluorescence detection of En1 (N,O) or Evx1 (P,Q) together with Phox2b (N-Q) on sections through r4 of wild-type (F,H,J,L,N,P,R) and Nkx6.1 mutant embryos (G,I,K,M,O,Q,S) at E10.5. These markers are similarly expressed in wild-type and in Nkx6.1 mutant embryos.